Quantitation of tamoxifen and metabolites thereof by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of tamoxifen and its metabolites in a sample by mass spectrometry. In some aspects, the methods provided herein determine the amount of norendoxifen. In some aspects, the methods provided herein determine the amount of norendoxifen and tamoxifen. In some aspects, the methods provided herein determine the amount of norendoxifen and other tamoxifen metabolites. In some aspects, the methods provided herein determine the amount of tamoxifen, norendoxifen, and other tamoxifen metabolites.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/992,214, filed May 12, 2014, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

Tamoxifen is a standard of treatment for hormone receptor positive breast cancer patients after primary treatment. Five to ten years of tamoxifen therapy reduces the risk of recurrence and death in these patients. Yet, many patients do not complete their full course of therapy, frequently due to unpleasant side effects of the drug.

Tamoxifen is a pro-drug which is converted to the highly active Endoxifen for full effectiveness. Conversion of Tamoxifen to Endoxifen is through a metabolic pathway dependent on genetic variation, such as that in CYP2D6 (2D6). Although 2D6 genotyping has been promoted to predict response to Tamoxifen and toxicity, the direct association on an individual level is controversial.

An effective method of predicting response to Tamoxifen is needed.

SUMMARY OF THE INVENTION

The present invention provides methods for quantitation of tamoxifen and its metabolites in a sample by mass spectrometry, including tandem mass spectrometry.

In one aspect, methods are provided for determining the amount of norendoxifen in a sample by mass spectrometry, comprising (a) ionizing said norendoxifen to produce one or more norendoxifen ions detectable by mass spectrometry; (b) detecting the amount of the norendoxifen ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of norendoxifen in said sample.

In one aspect, methods are provided for determining the amount of tamoxifen and metabolites thereof in a sample in a single mass spectrometry assay, comprising (a) ionizing said tamoxifen and metabolites to produce one or more ions detectable by mass spectrometry; (b) detecting the amount of the ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of each of tamoxifen and metabolites in said sample.

In some embodiments, said metabolites comprise norendoxifen. In some embodiments, said metabolites comprise endoxifen or N-Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethyl-4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethyl Tamoxifen. In some embodiments, said metabolites comprise metabolites selected from the group consisting of norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl-4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise any combination of norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl Tamoxifen. In some embodiments, said metabolites comprise norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl-4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise any combination of norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl Tamoxifen.

In one aspect, methods provided herein comprise protein precipitation. In some embodiments, methods provided herein comprise purification. In some embodiments, said purification comprises filtration. In some embodiments, said purification comprises liquid chromatography. In some embodiments, said liquid chromatography is high pressure liquid chromatography (HPLC).

In some embodiments, methods provided herein comprise detecting the amount of an internal standard. In some embodiments, said internal standard is a deuterated norendoxifen.

In some embodiments, ionization is by atmospheric pressure chemical ionization (APCI). In some embodiments, said ionization is in positive ion mode.

In some embodiments, ionization is by electrospray ionization (ESI). In some embodiments, said ionization is in positive ion mode.

In some embodiments, said sample is a serum sample.

In some embodiments, mass spectrometry is tandem mass spectrometry.

In one aspect, provided herein is a method for predicting tamoxifen response in a patient by determining the amount of tamoxifen or one or more tamoxifen metabolites. In some embodiments, a high amount of one or more tamoxifen or tamoxifen metabolites indicate a positive response to tamoxifen in a patient. In some embodiments, said metabolites comprise norendoxifen. In some embodiments, said metabolites comprise endoxifen or N-Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethyl-4′-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethyl Tamoxifen. In some embodiments, said metabolites comprise norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl-4′-Hydroxy Tamoxifen.

In some embodiments, the method provided herein has sensitivity measured by limit of quantitation (LOQ). In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.4 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL.

In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 0.5 ng/mL.

In some embodiments, the method provided herein has sensitivity measured by limit of detection (LOD). In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.15 ng/mL.

In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL.

In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 0.5 ng/mL.

As used herein, the term “purification” or “purifying” does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components of the sample. Purification, as used herein, does not require the isolation of an analyte from all others. In preferred embodiments, a purification step or procedure can be used to remove one or more interfering substances, e.g., one or more substances that would interfere with the operation of the instruments used in the methods or substances that may interfere with the detection of an analyte ion by mass spectrometry.

As used herein, the term “about” in reference to quantitative measurements, not including the measurement of mass of an ion, refers to the indicated value plus or minus 10%.

As used herein, the term “substantially all” refers to any proportion greater than 50%, more preferably greater than 60%, more preferably greater than 70%, more preferably greater than 80%, and more preferably greater than 90%.

As used herein, the term “sample” refers to any sample that may contain the analyte of interest. As used herein, the term “body fluid or tissue” means any fluid or tissue that can be isolated from the body of an individual. For example, “body fluid or tissue” may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like. If solid tissue is to be analyzed, it may be processed to release a liquid fraction that could contain any analyte present in the tissue. The liquid fraction can then be subject to the methods described herein.

As used herein, the term “size separation technique” means any technique (physical or chemical) that allows for the separation of at least one species from a test sample based on any one or more of molecular weight and shape. Examples of such techniques include, but are not limited to, filtration, chromatography, and certain aspects of mass spectrometry.

As used herein, the term “chromatography” refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around, over, and/or through a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). “Liquid chromatography” includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or “HPLC” refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their m/z. MS technology generally includes (1) ionizing the compounds to form charged species (e.g., ions); and (2) detecting the molecular weight of the ions and calculating their m/z. The compounds may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;” Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000).

As used herein, the term “operating in positive ion mode” refers to those mass spectrometry methods where positive ions are detected. Similarly, the term “operating in negative ion mode” refers to those mass spectrometry methods where negative ions are detected.

As used herein, the term “ionization” or “ionizing” refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Positive ions are those having a net positive charge of one or more electron units. Negative ions are those having a net negative charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization” or “MALDI” refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

As used herein, the term “surface enhanced laser desorption ionization” or “SELDI” refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or “APCI,” refers to mass spectroscopy methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N₂ gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

The term “Atmospheric Pressure Photoionization” or “APPI” as used herein refers to the form of mass spectroscopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins, A. P. (2000): See, e.g., Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography-mass spectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers to methods in which a sample is interacted with a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements

As used, herein, the term “field desorption” refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase.

As used herein, the term “limit of quantification” or “LOQ” refers to the point where measurements become quantitatively meaningful. The analyte response at this LOQ is identifiable, discrete and reproducible with a precision of 20% and an accuracy of 80% to 120%.

In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. In certain particularly preferred embodiments of the methods disclosed herein, mass spectrometry is performed using ESI.

In other preferred embodiments, a separately detectable internal standard is provided in the sample.

In one embodiment, the methods involve the combination of LC with mass spectrometry. In another preferred embodiment, the mass spectrometry is tandem mass spectrometry (MS/MS).

The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for quantitatively measuring tamoxifen and/or metabolites thereof in a patient sample. This quantitative measurement is achieved through the use of LC-MS/MS techniques. Prior to the use of LC-MS/MS, samples may be prepared by the following technique, or any portion thereof. A first purification of tamoxifen and/or metabolites thereof in a sample may be conducted through the use of a protein purification, filtration, or chromatography.

Any suitable size separation technique may be utilized, but in the examples that follow, both the first and second size separation techniques are filtration through a molecular weight cut-off filter. It is also possible, as discussed in the Examples that follow, to select a molecular weight cut-off filter with an appropriate molecular weight cut-off such that the same filter can be used for both the first size separation and the second size separation.

LC, most preferably HPLC, is utilized, may be utilized either alone or in combination with other purification methods, to purify selected analytes. This purification is combined with MS/MS, thereby providing an assay system for quantifying selected analytes in a test sample. The quantity of the selected analytes in the sample is then used to determine the quantity of tamoxifen in the original test sample. The quantitation methods provided herein have enhanced specificity and are less subject to methodological problems (such as antibody interference).

Suitable samples may include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, and the like. In certain preferred embodiments, samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably humans. Particularly preferred samples include blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid, or other body fluid or tissue samples. Such samples may be obtained, for example, from a patient; that is, a living person presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, serum or plasma.

Sample Preparation for Mass Spectrometry

Samples may be processed or purified to obtain preparations that are suitable for analysis by mass spectrometry. Such purification will usually include chromatography, such as liquid chromatography, and may also often involve an additional purification procedure that is performed prior to chromatography. Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, centrifugation, combinations thereof and the like.

Filtration is one preferred method of preparing a test sample, especially a biological test sample, such as serum or plasma, for chromatography. Such filtration is carried out by filtering a test sample through a molecular weight cut-off filter to separate species with molecular weights higher than the filter's cut-off from those with molecular weights lower than the filter's cut-off. The test sample remaining above the filter following complete (or near complete) filtration is substantially free of potentially interfering species with molecular weights lower than the filter's cut-off.

Various methods have been described involving the use of HPLC for sample clean-up prior to mass spectrometry analysis. See, e.g., Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a C18 analytical column, and MS/MS analysis); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a C18 analytical column, and MS/MS analysis). One of skill in the art may select HPLC instruments and columns that are suitable for use in the methods. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bonded alkyl groups, preferably C-8 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample.

In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. Such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

In one embodiment, the sample to be analyzed is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. In preferred embodiments, HPLC is performed on an analytical HPLC system with a C8 solid phase using 0.2% formic acid in HPLC Grade Ultra Pure Water and 0.2% formic acid in 100% methanol as the mobile phases.

Numerous column packings are available for chromatographic separation of samples and selection of an appropriate separation protocol is an empirical process that depends on the sample characteristics, analyte of interest, presence of interfering substances and their characteristics, etc. Commercially available HPLC columns include, but are not limited to, polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18, and polar coating on porous polymer columns.

In one embodiment, the HPLC column has a C8 solid phase with a median particle size of 5 μm (nominal) and a median particle pore size of 100 Å. In a preferred embodiment the column dimensions are 1.0 mm ID×50 mm length (Phenomenex Corp. Luna 5μ C8(2) 100 Å New Column 50×1.0 mm, Phenomenex Cat. No. 00B-4249-A0 or equivalent).

During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a “mobile phase”), choice of gradient elution and the gradient conditions, temperature, etc.

Detection and Quantitation by Mass Spectrometry

In various embodiments, analytes may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. Ionization sources used in various MS techniques include, but are not limited to, electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

In preferred embodiments, analytes are ionized by electrospray ionization (ESI) creating analyte precursor ions. In related preferred embodiments, analyte precursor ions are in a gaseous state and the inert collision gas is argon.

After the sample has been ionized, the positively charged ions thereby created may be analyzed to determine m/z. Suitable analyzers for determining m/z include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. The ions may be detected using one of several detection modes. For example, only selected ions may be detected using a selective ion monitoring mode (SIM), or alternatively, multiple ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). In preferred embodiments, ions are detected using SRM.

Preferably, m/z is determined using a quadrupole instrument. In a “quadrupole” or “quadrupole ion trap” instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and m/z. The voltage and amplitude may be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandem mass spectrometry,” or “MS/MS.” In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collision with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.

Additionally, recent advances in technology, such as matrix-assisted laser desorption ionization coupled with time-of-flight analyzers (“MALDI-TOF”) permit the analysis of analytes at femtomole levels in very short ion pulses. Mass spectrometers that combine time-of-flight analyzers with tandem MS are also well known to the artisan. Additionally, multiple mass spectrometry steps may be combined in methods known as “MS/MS”. Various other combinations may be employed, such as MS/MS/TOF, MALDI/MS/MS/TOF, or SELDI/MS/MS/TOF mass spectrometry.

The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each ion with a particular m/z over a given range (e.g., 400 to 1600 amu). The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, molecular standards may be run with the samples and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of tamoxifen. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the LC purification and mass spectrometry steps may be performed in an on-line fashion.

In certain embodiments, techniques such as MS/MS are used to isolate precursor ions for further fragmentation. In these embodiments, collision activation dissociation (CAD) may be used to generate the fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as “unimolecular decomposition”. Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy. In alternative embodiments, electron transfer dissociation (ETD) may be used to generate the fragment ions. In ETD, radical anions are used to transfer electrons to multiply charged peptide or protein cations resulting in random cleavage along the peptide backbone.

In particularly preferred embodiments, analyte is detected and/or quantified using LC-MS/MS as follows. An analyte enriched sample prepared as described above is subjected to LC. The flow of liquid solvent from the chromatographic column enters the heated nebulizer interface of a LC-MS/MS analyzer and the solvent/analyte mixture is converted to vapor in the heated tubing of the interface. The analyte contained in the nebulized solvent, is ionized by the corona discharge needle of the interface, which applies a large voltage to the nebulized solvent/analyte mixture. The ions pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e., “precursor” and “fragment” ions) based on their m/z. Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. Q1 selects for ions with m/z of precursor ions. Selected precursor ions are allowed to pass into the collision chamber (Q2), while ions with any other m/z collide with the sides of Q1 and are eliminated. Precursor ions entering Q2 may be fragmented with collision activated dissociation (CAD) through collisions with neutral argon gas molecules. Alternatively, if the precursor ions entering Q2 are multiply charged cations, they may be fragmented with electron transfer dissociation (ETD). The fragment ions generated are passed into Q3, where selected fragment ions are collected while other ions are eliminated.

Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion that may be used for selection in Q3. A specific fragment ion is one that will not be formed in significant amounts by other molecules with similar molecular structures. In contrast, a non-specific fragment ion is one that is formed by molecules other than the desired analyte. Suitable specific fragment ions can be identified by testing various molecular standards to determine whether fragment ions formed by a selected analyte are also formed by other molecules with similar structures or features. Preferably, at least one fragment ion specific for ions with m/z corresponding to that of analyte ions are identified.

As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots ion counts per unit time. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, are measured and the area or amplitude is correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of analytes with m/z. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard. The absolute amount of an analyte detected by LC-MS/MS can then be converted into an absolute amount of analyte that was present in the original test sample.

In some embodiments, the method provided herein has sensitivity measured by limit of quantitation (LOQ). In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.4 ng/mL.

In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL.

In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 0.5 ng/mL.

In some embodiments, the method provided herein has sensitivity measured by limit of detection (LOD). In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.15 ng/mL.

In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4′-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL.

In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 0.5 ng/mL.

The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Determination of Tamoxifen and its Metabolites

In the following procedure, tamoxifen and its metabolites are extracted from serum. The serum is added to a filter plate, and then mixed with acetonitrile/IS, which then forms a precipitate. The mixture is then placed on a positive pressure manifold, and the organic fraction is passed through to a collection plate. The plate is lidded, and then placed on the Cohesive system for injection onto MS/MS. The MS is in APCI-positive mode. The quantitation is based upon unique parent-product transitions. Those analytes with similar transitions were separated chromatographically.

Patient Preparation

Component Special Notations Fasting/Special Diets N/A Specimen Collection N/A and/or Timing Special Collection N/A Procedures Other No aromatase inhibitors.

Specimen Type & Handling

Criteria Type Preferred Serum Other Acceptable Collection Container Red top (no gel) Volume Optimum 0.5 mL - Optimum Minimum 0.3 mL - Minimum Transport Container & Frozen Temperature Stability & Storage Room Temperature: 5 Days Requirements Refrigerated: 7 Days Frozen: 31 Days Timing Considerations Unacceptable Plasma Samples Specimens & Actions Samples in SST tubes to Take Hemolyzed samples not acceptable. Compromising Physical Characteristics Other Considerations

Reagent Summary

Reagents Supplier & Catalog Number Quantity Ammonium Formate Sigma, 17843-250G or verified 250 Grams equivalent Formic Acid Sigma, F0507-100 mL or 100 mL verified equivalent Ethanol, Absolute, Pharmco-AAPER, 1 pint Anhydrous, 200 Proof Cat#111000200 or verified equivalent Acetonitrile, HPLC B&J, Cat #015-4 or verified 4 Liters Grade equivalent Biocell Serum, Human Biocell Labs, Cat # 1121-00 or 4 Liter Serum, Stripped and verified equivalent Delipidized (Opticlear) Tamoxifen Toronto Research Labs (TRC), 25 Grams Cat#T006000 or verified equivalent N-Desmethyl Toronto Research Labs (TRC), 50 mg Tamoxifen HCl Cat#D293900 or verified equivalent N-Desmethyl-4- Toronto Research Labs (TRC), 50 mg Hydroxy Tamoxifen Cat#D292043 or verified equivalent N-Desmethyl-4′- Toronto Research Labs (TRC), 10 mg Hydroxy Tamoxifen Cat#D292041 or verified equivalent (Z)-4-Hydroxy Toronto Research Labs (TRC), 100 mg Tamoxifen Cat#H954725 or verified equivalent 4′-Hydroxy Tamoxifen Toronto Research Labs (TRC), 25 mg Cat#H954730 or verified equivalent (E/Z)-Tamoxifen- Toronto Research Labs (TRC), 10 mg d5 Cat#T006007 or verified equivalent N-Desmethyl Toronto Research Labs (TRC), 10 mg Tamoxifen-d5 Cat#D293902 or verified equivalent N-Desmethyl-4- Toronto Research Labs (TRC), 10 mg Hydroxy Tamoxifen- Cat#D292044 or verified d5 equivalent N-Desmethyl-4′- Toronto Research Labs (TRC), 10 mg Hydroxy Tamoxifen- Cat#D291867 or verified d3 equivalent (Z)-4-Hydroxy Toronto Research Labs (TRC), 25 mg Tamoxifen-d5 Cat#H954757 or verified equivalent 4′-Hydroxy Toronto Research Labs (TRC), 10 mg Tamoxifen-d6 Cat#H954757 or verified equivalent

Calibrators/Standards Used

A 12-point calibration is used for each analyte. Initially, only one standard is made (Std-12), with a series of dilutions performed to generate the remaining standards. The standard (#12) is to be removed from the −70° C. freezer and thawed. While thawing, label twelve 12×75 mm tubes.

Add 3.0 mL of std-12 to tube 12. From this standard, you will follow the table below to create the standard curve. A standard curve is to be generated with each assay. Place the initial standard back in the −60 to −90° C. freezer.

Target Volume Analyte Concentration (ng/mL) to add (1 mg/mL) Tamoxifen 1,250 250 uL  N-Desmethyl Tamoxifen 1,250 250 uL  N-Desmethyl-4-Hydroxy 300 60 uL Tamoxifen (Endoxifen) N-Desmethyl-4′-Hydroxy 300 60 uL Tamoxifen 4-Hydroxy Tamoxifen 200 40 uL 4′-Hydroxy Tamoxifen 200 40 uL

Once these analytes have been added, QS to 200 mL with Biocell serum. Mix, then aliquot into 15 mL centrifuge tubes. Label (Std-12), then place the tubes in the −60° C. to −90° C. freezer for storage. Stable for 1 year.

Standard Target Concentrations

Target Target Target Concentration Target Concentration (N- Concen- (N-Desmethyl- Concentration Desmethyl tration 4′-Hydroxy Std # (Tamoxifen) Tamoxifen)) (Endoxifen) tamoxifen) 12 1250 1250 300 300 11 1000 1000 240 240 10 750 750 180 180 9 500 500 120 120 8 400 400 96 96 7 250 250 60 60 6 100 100 24 24 5 50 50 9 9 4 25 25 6 6 3 10 10 1.8 1.8 2 5 5 0.9 0.9 1 2.5 2.5 0.45 0.45 *Note: All concentrations in ng/mL.

Target Target Concentration(4- Concentration(4′- Std # Hydroxy Tamoxifen) Hydroxy Tamoxifen) 12 200 200 11 160 160 10 120 120 9 80 80 8 64 64 7 40 40 6 16 16 5 6 6 4 4 4 3 1.2 1.2 2 0.6 0.6 1 0.3 0.3

Calibration Curve Dilutions: Diluent (Biocell Std # Serum-mL) Std Added (mL) Std Used 12 0.0 As Needed 12 (Undiluted) 11 0.4 1.6 12 10 0.4 0.6 12 9 1.0 1.0 11 8 0.4 0.6 11 7 0.8 0.3 11 6 0.9 0.1 11 5 1.0 1.0 6 4 0.8 0.3 6 3 0.9 0.1 6 2 0.5 0.5 3 1 0.9 0.1 3

Calibration Procedure

Criteria Special Notations Frequency A calibration curve is to be set up every assay. Tolerance Limits Curve fit correlation must be 0.99 or greater. Procedure Refer to the dilution table in section 5.2. Freeze/Thaw studies were conducted, and showed that the analytes were stable over 5 cycles. Always return Std-12 to the −60° C. to −90° C. freezer as soon as you are finished with it. Use Quadratic curve fit for all analytes, except Endoxifen (linear). Origin's are set to Ignore, and weighting is set to 1/y.

Equipment and Supplies

Assay Platform

A Thermo LC/MS/MS system containing the following modules was used for this assay:

Modules Thermo Quantum Ultra (Serial #022-TQU01376) Cohesive Aria TLX-4 (Serial # SJCTX457) LC Quant Software (Xcalibur, Thermo Fisher) Aria 1.6 Software Agilent G1312 Binary Pumps Thermo APCI Source

Equipment

Equipment Supplier Cat# SPE Positive Pressure Manifold SPEWare Corp BDS Hypersil C18, 150 × 4.6, 5u Thermo Scientific P/N 28105-154630 Eppindorf Repeater Pipette Eppindorf/Cardinal P5063-20 Health Gilson P-200 Pipette Gilson Gilson P-1000 Pipette Gilson 20 mL Scintillation Vials Warehouse Stock Clerk w/white cap # 128623VX- 2500 Multi-Tube Vortexer VWR/SP

Supplies

Supply Supplier Cat # 250 uL Rainen Pipette Tips Rainen HR-250 1000 uL Pipette Tips Rainen HR-1000 Thermo 96 Deep Well Plates Thermo Scientific P/N 260252 Thermo Pre-Slit Well Cap for 96 Thermo Scientific P/N 276011 Well PP Plate Sirocco Protein Precipitation Plate, Waters Corp P/N 186002448 5/pk

Mass Spectrometry

Retention Time Parent Analyte (RT) Expected Ion Product Ion Tamoxifen 4.93 372.2 72.15 E/Z Tamoxifen-d5 4.42(E), 4.90(Z)* 377.2 72.18 N-Desmethyl Tamoxifen 4.65 358.2 58.15 N-Desmethyl Tamoxifen-d5 4.62 363.2 58.13 4-Hydroxy Tamoxifen 1.68 388.2 72.15 (Z)-4-Hydroxy Tamoxifen-d5 1.64 393.2 72.15 4′-Hydroxy Tamoxifen 2.86 388.2 72.15 4′-Hydroxy Tamoxifen-d6 2.84 394.2 78.18 N-Desmethyl-4-Hydroxy 1.16 374.2 58.15 Tamoxifen (Endoxifen) N-Desmethyl-4-Hydroxy 1.16 379.24 58.15 Tamoxifen-d5 N-Desmethyl-4′-Hydroxy 2.40 374.2 58.15 Tamoxifen N-Desmethyl-4′Hydroxy 2.38 377.2 61.20 Tamoxifen-d3 *The Z-form is the active form that is monitored.

Expected Values

Reference Ranges:

Tamoxifen: 12.54-233.07 ng/mL

N-Desmethyl Tamoxifen: 2.59-373.96 ng/mL

4′-Hydroxy Tamoxifen: 0.4-6.33 ng/mL

4-Hydroxy Tamoxifen: 0.24-5.05 ng/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen): 0.93-43.19 ng/mL

N-Desmethyl-4′-Hydroxy Tamoxifen: 1.17-19.95 ng/mL

Analytical Measurement Range (AMR)

Tamoxifen: 1.47-1500 ng/mL

N-Desmethyl Tamoxifen: 1.47-1500 ng/mL

4′-Hydroxy Tamoxifen: 0.2-200 ng/mL

4-Hydroxy Tamoxifen: 0.2-200 ng/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen): 0.39-400 ng/mL

N-Desmethyl-4′-Hydroxy Tamoxifen: 0.39-400 ng/mL

Precision: Inter-(span of assay) and intra-assay (1 day) precision studies were performed using the low, medium, and high controls. All analytes showed a <12% CV over the span of the validation.

Interfering Substances: Mild or moderate icteric and lipemic samples are acceptable. Hemolytic samples are not acceptable, as they will clog the filter during sample prep. Grossly hemolyzed, icteric, and lipemic samples are not acceptable.

Clinical Sensitivity (LOQ):

Tamoxifen: 1.47 ng/mL

N-Desmethyl Tamoxifen: 1.46 ng/mL

4′-Hydroxy Tamoxifen: 0.2 ng/mL

4-Hydroxy Tamoxifen: 0.2 ng/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen): 0.39 ng/mL

N-Desmethyl-4′-Hydroxy Tamoxifen: 0.39 ng/mL

Example 2 Validation of Tamoxifen and its Metabolites Assay

This report contains a detailed summary of the validation for Tamoxifen and its 5 main phase I metabolites by LC/MS/MS. The assay is a laboratory-developed test.

Methodology: Tamoxifen and its five main phase I metabolites (N-desmethyltamoxifen, N-Desmethyl-4-hydroxytamoxifen, N-desmehtyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen, and 4′-hydroxytamoxifen) are extracted from serum. The extraction is a protein precipitation, followed by filtration. Analysis and quantitation is then performed by LC/MS/MS.

Kit or Reagents

Reagents Supplier & Catalog Number Ammonium Formate Sigma, 17843-250G Formic Acid Sigma, F0507-100 mL Ethanol, Absolute, Anhydrous, Pharmco-AAPER, 200 Proof Cat#111000200 Acetonitrile, HPLC Grade B&J, Cat #015-4 Biocell Serum, Human Serum, Stripped Biocell Labs, Cat # 1121-00 and Delipidized (Opticlear) Tamoxifen Toronto Research Labs (TRC), Cat#T006000 N-Desmethyl Tamoxifen HCl Toronto Research Labs (TRC), Cat#D293900 N-Desmethyl-4-Hydroxy Tamoxifen Toronto Research Labs (TRC), Cat#D292043 N-Desmethyl-4′-Hydroxy Tamoxifen Toronto Research Labs (TRC), Cat#D292041 (Z)-4-Hydroxy Tamoxifen Toronto Research Labs (TRC), Cat#H954725 4′-Hydroxy Tamoxifen Toronto Research Labs (TRC), Cat#H954730 (E/Z)-Tamoxifen-d5 Toronto Research Labs (TRC), Cat#T006007 N-Desmethyl Tamoxifen-d5 Toronto Research Labs (TRC), Cat#D293902 N-Desmethyl-4-Hydroxy Tamoxifen-d5 Toronto Research Labs (TRC), Cat#D292044 N-Desmethyl-4′-Hydroxy Tamoxifen-d3 Toronto Research Labs (TRC), Cat#D291867 (Z)-4-Hydroxy Tamoxifen-d5 Toronto Research Labs (TRC), Cat#H954757 4′-Hydroxy Tamoxifen-d6 Toronto Research Labs (TRC), Cat#H954757

Precision Study for Laboratory Developed Tests (LDT)

Within Run Precision:Low, medium, and high controls were analyzed (10) within one run. All QC fell within acceptability criteria (<20% CV).

4-OH- N-DM-4′OH- 4′OH- N-DM- Endoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) Low Control 1 11.479 1.582 10.170 1.926 34.728 32.814 2 7.510 1.806 8.915 1.757 32.952 39.576 3 8.580 1.751 9.064 1.552 36.389 37.948 4 9.156 1.833 9.196 1.641 35.488 33.814 5 9.805 1.844 9.221 1.643 33.879 31.212 6 9.088 2.132 8.785 1.758 32.435 34.300 7 8.821 2.079 8.569 1.559 33.555 36.784 8 7.304 2.383 8.440 1.994 35.117 33.327 9 9.921 2.119 9.136 1.745 37.190 33.794 10 8.550 2.192 8.102 1.845 38.720 36.350 Mean 9.02 1.97 8.96 1.74 35.05 34.99 Stdev 1.21 0.24 0.56 0.15 1.97 2.58 % CV 13.41 12.42 6.25 8.51 5.63 7.37 Middle Control 1 83.799 39.593 80.603 34.332 165.439 186.498 2 88.134 37.585 79.798 36.082 181.275 205.514 3 84.445 35.894 77.389 36.070 164.816 172.721 4 83.364 36.412 83.648 34.483 164.643 174.957 5 82.632 35.697 83.397 35.866 176.518 177.587 6 88.988 37.536 75.551 31.615 170.129 182.202 7 85.351 38.475 80.059 35.940 162.261 198.626 8 81.834 38.402 77.803 32.461 176.554 184.688 9 86.598 39.946 84.175 35.302 163.246 204.461 10 95.135 37.530 79.668 37.393 196.225 187.704 Mean 86.03 37.71 80.21 34.95 172.11 187.50 Stdev 3.95 1.44 2.86 1.78 10.74 11.77 % CV 4.59 3.83 3.57 5.08 6.24 6.28 High Control 1 189.670 93.480 188.415 92.408 658.605 858.395 2 201.279 91.872 189.325 91.608 755.094 882.797 3 189.009 95.969 174.819 84.116 724.537 780.958 4 201.229 92.422 190.193 87.989 719.015 887.024 5 198.358 88.508 181.242 92.937 683.269 763.216 6 170.784 85.174 177.770 85.490 721.171 823.250 7 184.641 98.301 172.371 86.206 706.818 783.975 8 205.803 89.703 184.633 84.096 712.341 784.042 9 190.205 94.532 164.908 84.977 723.057 805.741 10 190.731 89.605 189.710 96.315 737.294 819.099 Mean 192.17 91.96 181.34 88.61 714.12 818.85 Stdev 10.14 3.86 8.68 4.36 27.01 44.03 % CV 5.28 4.20 4.79 4.92 3.78 5.38

Total Precision:

Total precision was based on all QC run on all assays during the validation process. Acceptability was based on <20% CV. All QC fell within this criteria.

4-OH- N-DM-4′OH- 4′OH- N-DM- Endoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) Low Control 1 11.592 2.285 8.663 1.525 36.145 35.525 2 11.138 2.277 9.762 2.168 40.870 38.152 3 8.890 2.519 10.094 2.145 40.451 37.248 4 10.022 2.417 10.096 2.363 34.538 38.348 5 10.377 1.964 9.531 2.130 40.335 37.198 1 8.917 2.169 8.172 1.932 34.775 35.754 2 9.833 1.926 9.707 1.953 37.485 34.955 3 8.974 2.066 9.451 2.239 36.460 36.176 4 9.810 2.035 8.760 1.621 32.040 32.630 5 10.367 1.751 8.983 1.847 36.114 37.263 1 12.282 2.410 8.462 2.106 34.382 33.980 2 8.801 2.418 9.214 1.696 38.913 37.524 3 10.512 1.650 9.246 2.038 35.886 39.920 4 9.479 2.072 8.300 1.925 40.630 40.013 5 9.487 2.094 9.062 2.260 35.468 40.544 1 13.192 2.172 9.722 2.067 34.702 36.737 2 11.281 1.876 9.876 1.723 31.707 36.750 3 9.727 1.778 9.547 2.085 38.602 34.458 4 10.920 1.956 9.623 1.902 35.276 36.427 5 10.765 1.878 10.720 2.196 32.678 37.328 1 9.905 2.267 9.573 1.756 34.575 40.865 2 9.517 2.419 10.397 1.852 38.703 38.931 3 10.592 2.089 10.440 1.768 36.104 39.576 4 9.705 2.161 10.924 1.881 37.768 40.039 5 9.299 1.964 9.492 1.660 35.499 37.825 1 10.312 2.195 11.309 1.952 37.109 37.409 2 8.540 2.084 10.329 1.848 32.896 39.378 1 9.971 2.699 10.214 1.968 37.176 38.498 2 10.845 2.264 8.237 1.985 38.033 42.613 3 9.275 2.184 8.027 1.590 32.633 34.847 4 7.925 2.569 7.925 2.166 35.327 35.302 5 8.041 2.312 7.885 1.891 31.681 36.267 1 8.636 2.075 9.928 2.023 34.570 38.068 2 10.653 2.122 10.316 2.038 39.078 38.219 1 10.003 2.559 9.009 2.401 41.780 38.067 2 13.146 2.143 9.527 2.285 37.945 36.085 1 9.319 2.202 9.477 2.347 34.554 38.110 2 8.029 1.836 8.287 1.789 33.201 30.038 3 9.012 2.140 8.720 1.974 35.778 34.251 4 8.299 2.037 9.456 2.119 38.542 37.302 1 9.606 2.449 10.127 2.619 38.680 42.268 2 11.108 2.410 9.118 2.570 38.456 42.861 3 11.502 2.288 8.941 2.224 36.295 39.003 4 9.983 2.536 9.583 2.301 39.444 42.359 1 10.863 2.027 9.713 1.955 39.137 36.636 2 9.912 2.046 8.556 2.133 35.665 38.773 1 9.603 2.247 7.986 2.063 42.388 39.865 2 9.305 2.071 9.373 2.197 37.447 39.204 1 8.518 2.203 9.189 2.097 36.600 34.954 2 8.727 2.108 8.134 1.868 33.344 33.870 3 9.676 2.242 8.135 2.234 34.319 35.267 4 8.867 2.041 7.973 2.008 36.328 36.998 1 8.988 2.006 10.200 2.013 37.349 36.239 2 9.274 1.870 9.697 1.967 38.264 38.313 1 9.503 1.919 9.453 2.068 43.706 33.935 2 9.703 2.007 8.722 1.874 40.296 34.916 Mean 9.87 2.15 9.31 2.03 36.75 37.39 Std Dev 1.15 0.22 0.83 0.23 2.77 2.55 CV 11.65 10.36 8.96 11.29 7.53 6.82 Middle Control 1 87.010 39.746 89.637 42.407 202.496 210.097 2 94.595 39.218 101.461 43.252 220.163 203.365 3 96.338 41.161 103.091 41.130 210.035 237.872 4 100.952 40.926 97.656 45.079 194.037 203.374 5 92.546 42.034 90.246 41.105 180.896 234.350 1 86.522 38.422 82.806 37.133 170.911 189.278 2 86.982 35.883 89.719 35.175 182.954 200.267 3 94.330 37.402 83.686 38.187 187.342 186.339 4 86.088 37.256 90.762 37.099 177.586 167.783 5 90.805 36.694 88.020 37.999 186.762 176.707 1 88.741 38.437 77.735 34.791 188.429 170.437 2 93.659 38.068 86.365 37.361 182.615 185.327 3 89.770 39.654 76.566 33.949 184.621 210.695 4 94.247 38.336 80.834 35.738 182.464 201.245 5 88.359 39.617 79.947 38.285 196.110 185.179 1 96.013 38.875 94.650 38.235 192.418 178.464 2 89.914 35.280 74.974 34.501 195.061 174.347 3 90.408 36.250 94.097 36.874 192.154 180.649 4 86.933 35.653 88.543 37.264 198.757 172.231 5 105.603 37.055 96.923 39.572 166.573 188.035 1 96.715 38.736 91.402 38.503 193.668 192.852 2 85.550 38.719 91.593 38.211 192.601 203.520 3 96.364 40.895 97.744 40.532 203.027 210.815 4 89.677 39.397 77.862 36.283 190.794 191.809 5 84.419 36.150 87.890 36.258 203.371 192.906 1 87.929 38.550 88.816 36.835 181.353 197.693 2 74.719 36.294 87.041 34.933 179.365 190.056 1 83.567 36.801 83.282 39.022 177.547 177.361 2 75.551 33.187 85.481 34.572 153.055 159.856 3 87.804 38.901 71.354 28.960 166.592 192.261 4 82.223 37.510 72.808 29.301 166.157 179.546 5 85.341 39.784 71.964 30.776 171.216 205.388 1 88.004 33.537 90.680 33.157 195.955 187.743 2 86.158 39.113 89.815 35.488 184.322 189.194 1 88.728 41.774 76.301 35.150 179.239 217.475 2 100.988 37.906 80.538 36.629 197.328 202.671 1 84.292 35.389 90.935 35.702 184.883 189.455 2 88.520 34.514 95.314 35.561 183.190 172.592 3 83.439 37.285 95.351 37.955 180.218 173.995 4 86.701 37.091 88.160 36.253 192.596 180.127 1 86.049 37.984 83.921 36.871 172.997 224.122 2 91.295 39.390 92.291 39.164 185.491 202.409 3 88.654 38.989 85.121 38.139 177.713 213.566 4 89.973 40.718 93.456 39.341 197.407 215.637 1 93.239 42.549 91.506 38.826 181.470 205.651 2 98.907 38.658 82.931 36.807 198.912 216.152 1 93.912 38.063 89.926 35.099 188.095 222.914 2 83.242 34.984 88.570 37.402 200.103 210.098 1 96.655 38.764 81.895 36.921 173.766 179.647 2 93.507 36.178 87.743 37.688 169.649 196.749 3 97.061 39.966 86.633 39.299 174.694 195.119 4 97.760 41.871 92.541 40.805 198.256 192.921 1 88.496 38.325 87.354 37.319 189.199 184.837 2 85.028 38.332 87.345 38.125 177.596 193.024 1 86.758 38.894 88.755 37.777 198.350 197.001 2 87.200 36.649 86.536 37.594 205.277 185.319 Mean 89.90 38.18 87.30 37.19 186.75 194.62 Std Dev 5.88 2.08 7.06 2.91 12.48 16.69 CV 6.54 5.44 8.08 7.82 6.68 8.58 High Control 1 207.479 101.732 195.254 102.024 799.588 735.969 2 190.387 102.016 226.236 108.850 860.365 798.839 3 202.213 98.424 199.550 101.300 856.971 783.327 4 210.429 102.722 202.273 106.471 811.375 829.711 5 212.515 109.031 222.061 111.194 857.859 859.031 1 196.747 103.011 204.676 99.578 816.049 747.992 2 206.775 95.510 180.867 87.664 710.089 875.109 3 186.389 95.141 195.533 88.284 671.914 798.004 4 184.079 93.074 170.879 85.515 680.514 748.155 5 196.620 93.918 166.410 91.662 696.733 813.829 1 181.815 101.602 205.865 92.189 786.902 889.986 2 204.310 94.568 194.635 92.144 823.327 787.045 3 203.649 95.490 182.781 85.872 790.504 798.055 4 205.176 92.576 189.464 89.338 758.539 768.107 5 202.342 96.793 176.749 92.032 846.377 727.994 1 198.640 98.625 196.187 95.798 810.498 728.474 2 198.899 91.019 187.599 100.136 751.812 805.304 3 199.035 84.927 179.312 95.215 737.664 770.813 4 212.276 84.154 190.044 100.630 734.151 886.035 5 200.109 92.086 194.237 99.469 760.246 872.936 1 200.469 89.448 199.269 91.661 729.412 834.619 2 202.977 98.208 199.034 96.375 790.310 799.638 3 205.475 93.973 192.105 92.720 798.244 810.004 4 197.479 99.860 196.147 94.396 761.732 783.978 5 194.612 94.240 197.186 97.771 786.241 771.571 1 186.505 81.241 198.300 87.311 710.289 749.671 2 176.188 89.968 181.780 79.731 731.985 741.325 1 199.356 87.775 207.198 101.803 748.141 771.557 2 222.589 105.058 196.074 90.785 801.007 942.577 3 181.161 94.362 148.799 78.682 617.203 695.000 4 215.118 100.450 158.172 79.628 750.937 839.526 5 191.211 102.290 163.134 79.661 725.107 704.289 1 196.775 98.926 191.427 91.397 676.187 806.089 2 218.733 94.497 188.927 83.249 702.543 764.559 1 189.423 103.108 176.834 85.740 787.894 783.972 2 203.993 104.811 242.863 100.905 744.233 775.926 1 200.471 95.865 198.445 89.203 766.594 768.276 2 202.665 90.224 209.309 97.688 728.383 781.862 3 204.180 101.425 203.688 92.536 677.014 860.784 4 199.399 86.145 187.369 82.171 719.400 701.285 1 197.465 92.884 200.642 95.198 736.030 826.979 2 190.613 93.842 183.426 97.415 744.250 800.504 3 199.096 96.602 193.312 96.739 746.794 776.233 4 179.140 87.981 197.976 93.276 691.001 866.107 1 199.248 95.517 205.052 89.399 718.152 830.034 2 217.882 94.943 190.257 89.531 720.014 762.305 1 200.190 93.715 197.387 100.663 748.026 831.065 2 213.258 94.615 190.476 91.976 762.683 800.202 1 185.724 96.274 187.941 94.235 707.147 798.423 2 217.069 101.477 191.859 96.905 823.232 803.716 3 213.982 95.430 181.301 98.054 700.987 770.842 4 199.874 92.896 172.759 92.304 735.885 814.102 1 182.741 98.602 185.285 95.342 715.912 774.003 2 182.816 90.468 216.778 95.733 747.020 861.042 1 179.231 95.177 187.040 88.321 748.849 740.018 2 198.872 89.425 194.280 92.437 758.636 851.201 Mean 199.03 95.50 192.37 93.33 752.12 796.75 Std Dev 11.02 5.62 15.76 7.07 51.25 50.69 CV 5.53 5.89 8.19 7.57 6.81 6.36

Analytical Sensitivity (Detection Limits)

Limit of Detection (LOD): Limit of detection (LOD) was performed by taking a low pool (containing all analytes), then serially diluting (1:2) down to the lowest observable level. Assuming the linearity were to continue below the level of quantitation (LOQ), the values below would be the lowest quantifiable concentrations. This experiment was performed over 5 days.

-   -   Tamoxifen=0.59 ng/mL     -   N-Desmethyl Tamoxifen=0.59 ng/mL     -   4′-Hydroxy Tamoxifen=0.1 ng/mL     -   N-Desmethyl-4′-Hydroxy Tamoxifen=0.5 ng/mL     -   4-Hydroxy Tamoxifen=0.1 ng/mL     -   N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen)=0.15 ng/mL

Limit of Quantitation (LOQ): The acceptability criteria for the LOQ is defined as the lowest concentration at which CV<20%. To determine the LOQ, a mid-level standard was diluted down serially 1:2.

Tamoxifen=1.5 ng/mL

N-Desmethyl Tamoxifen=1.5 ng/mL

4′-Hydroxy Tamoxifen=0.4 ng/mL

N-Desmethyl-4′-Hydroxy Tamoxifen=0.4 ng/mL

4-Hydroxy Tamoxifen=0.2 ng/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen)=0.4 ng/mL

Accuracy

Recovery of Known Standards:

Serum was spiked with all analytes to specific concentrations, extracted, and then analyzed in triplicate. All mixes were spiked to cover the linear and therapeutic range of each analyte.

Specified Calc. Conc. (ng/mL) Conc. (ng/mL) 1 2 3 Mean Std Dev CV % Recov Mix1 Endoxifen 10 9.52 10.20 10.96 10.23 0.72 7.04 102.27 4OH Tamoxifen 200 202.34 200.34 217.89 206.86 9.61 4.64 103.43 N-DM 4′ OH Tam 10 10.03 9.19 8.82 9.35 0.62 6.61 93.50 4′ OH Tamoxifen 200 188.92 180.49 220.38 196.60 21.02 10.69 98.30 N-DM Tamoxifen 500 456.99 433.11 469.66 453.26 18.56 4.09 90.65 Tamoxifen 500 474.32 464.31 498.06 478.90 17.34 3.62 95.78 Mean: 97.32 Mix2 Endoxifen 200 188.88 186.97 164.95 180.27 13.30 7.38 90.14 4OH Tamoxifen 5 4.16 4.73 4.38 4.42 0.29 6.48 88.49 N-DM 4′ OH Tam 200 204.90 190.67 178.00 191.20 13.45 7.04 95.60 4′ OH Tamoxifen 5 4.90 4.54 4.92 4.79 0.21 4.45 95.82 N-DM Tamoxifen 10 9.30 9.57 9.93 9.61 0.32 3.30 96.06 Tamoxifen 10 10.03 9.18 9.88 9.70 0.45 4.67 97.00 Mean: 93.85 Mix3 Endoxifen 100 87.92 89.91 91.72 89.85 1.90 2.11 89.85 4OH Tamoxifen 100 101.75 104.22 92.28 99.42 6.30 6.34 99.42 N-DM 4′ OH Tam 100 98.83 91.81 92.09 94.25 3.97 4.22 94.25 4′ OH Tamoxifen 100 93.10 90.39 87.88 90.46 2.61 2.89 90.46 N-DM Tamoxifen 1000 885.57 857.60 932.56 891.92 37.88 4.25 89.19 Tamoxifen 1000 994.90 908.44 870.80 924.72 63.63 6.88 92.47 Mean: 92.61 Mix4 Endoxifen 80 74.71 73.65 75.85 74.74 1.10 1.47 93.43 4OH Tamoxifen 10 7.74 10.21 9.77 9.25 1.32 14.27 92.47 N-DM 4′ OH Tam 80 78.97 73.72 70.50 74.40 4.28 5.75 93.00 4′ OH Tamoxifen 10 10.09 9.27 9.04 9.47 0.55 5.85 94.70 N-DM Tamoxifen 100 93.31 88.07 92.50 91.30 2.82 3.09 91.30 Tamoxifen 100 97.94 107.41 99.07 101.48 5.17 5.10 101.48 Mean: 94.40

Interference Study

Acceptability criteria: The difference due to a potential interfering substance should be ≦TEa/4 to be considered acceptable

Hemolysis Interference: Low and high pools were spiked with a hemolyzed RBC's at low, medium, and high concentrations. Samples were extracted in quadruplicate. Hemolysis showed no interference with tamoxifen or measured metabolites. However, due to difficulty with filtration from moderate and high hemolysis samples, only mildly hemolyzed samples should be accepted.

Low Medium High Hemolysis Low Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 109.28 110.30 115.24 (Endoxifen) 4-OH-Tamoxifen 106.99 108.40 104.81 N-Desmethyl-4′OH Tamoxifen 96.42 105.05 97.57 4′OH-Tamoxifen 102.37 99.02 108.24 N-DM-Tamoxifen 93.52 98.47 91.98 Tamoxifen 101.85 99.82 99.73 High Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 97.73 97.46 93.16 (Endoxifen) 4-OH-Tamoxifen 95.75 95.78 91.03 N-Desmethyl-4′OH Tamoxifen 98.33 95.33 95.39 4′OH-Tamoxifen 99.52 101.43 103.19 N-DM-Tamoxifen 92.42 95.50 93.45 Tamoxifen 91.51 95.13 105.25

Lipemia Interference: Low and high pools were spiked with a lipemic samples at low, medium, and high concentrations. Samples were extracted in quadruplicate. Lipemic samples showed no interference with tamoxifen or measured metabolites.

Low Medium High Lipemic Low Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 109.27 104.58 109.12 (Endoxifen) 4-OH-Tamoxifen 113.91 104.18 113.29 N-Desmethyl-4′OH Tamoxifen 102.52 103.49 113.76 4′OH-Tamoxifen 106.35 93.43 98.35 N-DM-Tamoxifen 97.89 97.34 90.64 Tamoxifen 101.74 95.64 103.72 High Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 97.21 99.49 96.19 (Endoxifen) 4-OH-Tamoxifen 94.63 98.42 94.17 N-Desmethyl-4′OH Tamoxifen 99.16 97.77 95.75 4′OH-Tamoxifen 101.37 103.79 101.82 N-DM-Tamoxifen 91.19 94.40 102.11 Tamoxifen 87.03 96.82 94.34

Bilirubin Interference: Low and high pools were spiked with a bilirubin at low, medium, and high concentrations. Samples were extracted in quadruplicate. Bilirubin spiked samples showed no interference with tamoxifen ormeasured metabolites.

Low Medium High Bilirubin Low Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 91.69 110.76 100.67 (Endoxifen) 4-OH-Tamoxifen 114.79 102.57 110.50 N-Desmethyl-4′OH Tamoxifen 107.02 103.14 100.46 4′OH-Tamoxifen 91.05 96.49 103.59 N-DM-Tamoxifen 89.89 100.83 91.15 Tamoxifen 97.04 103.01 96.17 High Pool (% Recovery) N-Desmethyl-4-Hydroxy Tamoxifen 103.15 97.78 95.72 (Endoxifen) 4-OH-Tamoxifen 98.80 97.99 89.94 N-Desmethyl-4′OH Tamoxifen 97.68 95.41 93.89 4′OH-Tamoxifen 101.44 100.35 99.97 N-DM-Tamoxifen 93.84 100.36 95.32 Tamoxifen 94.63 92.75 94.80

Example 3 Validation of Norendoxifen Assay

This report contains a detailed summary of the validation for Norendoxifen by LC/MS/MS. The assay is a laboratory-developed test.

Norendoxifen is extracted from serum using protein precipitation, followed by filtration. Analysis and quantitation is then performed by LC/MS/MS.

Kit or Reagents

Reagents Supplier & Catalog Number Ammonium Formate Sigma, 17843-250G Formic Acid Sigma, F0507-100 mL Ethanol, Absolute, Anhydrous, Pharmco-AAPER, 200 Proof Cat#111000200 Acetonitrile, HPLC Grade B&J, Cat #015-4 Biocell Serum, Human Serum, Stripped Biocell Labs, Cat # 1121-00 and Delipidized (Opticlear) Norendoxifen Dr. David Flockhart N-Desmethyl-4-Hydroxy Tamoxifen-d5 Toronto Research Labs (TRC), Cat#D292044

Precision Study for Laboratory Developed Tests (LDT)

Within Run Precision:Low, medium, and high controls (ng/mL) were analyzed (10) within one run. All QC fell within acceptability criteria (<20% CV).

Controls Low Mid High 1 8.71 103.75 188.84 2 8.35 104.13 195.45 3 10.65 92.25 211.45 4 9.42 95.71 197.29 5 7.66 93.26 175.46 6 9.95 88.97 156.57 7 8.65 111.42 152.57 8 8.00 93.23 172.76 9 9.66 84.69 178.21 10  8.76 92.88 172.45 mean 8.98 96.03 180.10 std dev 0.93 8.03 18.38 % cv 10.30 8.36 10.20

Total Precision:

Total precision was based on all QC run on all assays during the validation process. Acceptability was based on <20% CV. All QC fell within this criteria.

Limit of Detection (LOD): Norendoxifen=1.2 ng/mL

Limit of Quantitation (LOQ): Norendoxifen=1.2 ng/mL

Accuracy:

Recovery of Known Standards: Serum was spiked with all analytes to specific concentrations, extracted, and then analyzed in quadruplicate. All mixes were spiked to cover the linear and therapeutic range of each analyte.

Exp. Calculated Concentrations (ng/mL) Mix Conc 1 2 3 4 Mean Std Dev CV % Recov 1 10.00 10.67 10.53 10.27 9.04 10.13 0.75 7.37 101.26 2 20.00 25.49 21.84 19.60 21.83 22.19 2.44 11.01 110.95 3 110.00 108.48 110.16 117.29 102.75 109.67 5.99 5.46 99.70 4 150.00 150.74 154.81 146.46 164.19 154.05 7.57 4.91 102.70 5 200.00 202.59 185.39 192.42 170.60 187.75 13.44 7.16 93.87 6 40.00 40.40 41.99 38.88 39.59 40.22 1.33 3.32 100.54

Interference Study

Acceptability criteria: The difference due to a potential interfering substance should be ≦TEa/4 to be considered acceptable

Hemolysis Interference: Low and high pools were spiked with a hemolyzed RBC's at low, medium, and high concentrations. Samples were extracted in quadruplicate. Hemolysis showed no interference with norendoxifen. However, due to difficulty with filtration from moderate and high hemolyzed samples, only mildly hemolyzed samples should be accepted.

% Recovery Hemolysis Low Mid High Low Pool 98.80 109.49 107.30 High Pool 101.58 112.14 114.22

Lipemia Interference: Low and high pools were spiked with a lipemic samples at low, medium, and high concentrations. Samples were extracted in quadruplicate. Lipemic samples showed no interference with norendoxifen. Recovery of Known Standards: Serum was spiked with all analytes to specific concentrations, extracted, and then analyzed in quadruplicate. All mixes were spiked to cover the linear and therapeutic range of each analyte.

% Recovery Lipemic Low Mid High Low Pool 99.88 102.19 97.92 High Pool 100.76 96.05 92.19

Bilirubin Interference: Low and high pools were spiked with a bilirubin at low, medium, and high concentrations. Samples were extracted in quadruplicate. Bilirubin spiked samples showed no interference with norendoxifen.

% Recovery Icterus Low Mid High Low Pool 100.42 98.45 100.92 High Pool 95.63 96.76 94.44

Example 4 Clinical Quantitation and Response Study

The standard operating protocol of Examples 1-3 were used to quantitate tamoxifen and its metabolites in patient samples and were correlated to tamoxifen response.

Norendoxifen Quantitation and Response in Patient Samples.

Norendoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.25 4733 948878 18788 1.34 9020 764437 18824 0.88 7074 793359 18837 1.43 7993 645545 18865 1.74 11233 784027 18873 2.02 13715 851690 18885 1.53 18676 1434599 20434 0.14 6485 1524835 20478 0.35 10472 1873595 20484 0.44 8693 1411744 20511 1.55 19241 1466708 20515 0.82 11924 1392281 20564 2.38 27537 1498317 20573 1.63 19951 1461115 20581 3.33 34700 1426339 20601 3.22 37814 1599850 20647 4.45 46786 1490262 20611 3.25 33755 1415658 20689 4.36 44265 1436705 20704 4.62 47340 1458681 26616 0.48 9695 1508141 26690 1.03 14558 1475191 26756 1.25 16288 1447066 26777 2.02 20735 1287689 26801 1.70 18633 1323903 26826 2.12 16022 959275 26837 0.24 5498 1115449 26853 2.51 18098 944719 26864 1.13 8617 822666 26878 2.40 14663 794035 26939 1.38 9809 812995 26952 1.23 9836 886924 26958 0.95 6946 744284 26978 1.67 12349 887541 27009 1.15 8821 828349 27011 2.49 16676 876309 27022 1.23 7675 689142 27045 1.34 8099 686202 27067 −0.16 1477 615842 27080 0.19 3606 785747 27084 0.31 4276 798627 27413 1.04 4611 465635 27446 −0.32 883 630981 27482 1.33 7572 643410 27526 2.78 12639 606397 27539 1.72 8159 574207 30002 0.26 2344 466484 30031 0.89 4453 495349 30043 0.51 3321 505290 30079 3.53 12938 505353 30105 1.54 9701 743460 30116 0.92 6705 731395 30127 1.90 9658 629546 6420 0.41 6415 1070269 6482 1.61 15116 1121187 6497 1.40 14181 1162006 6510 0.73 6127 767435 6521 1.68 9575 687725 6535 1.03 8685 882954 6549 3.54 25094 978851   1 1.22 11722 1062595   2 2.22 18544 1069320   3 4.61 36683 1132746   4 17.07 117804 1056612   5 11.63 90103 1172890   6 23.61 178911 1166491   7 60.70 453821 1145572   8 99.24 751764 1143017   9 119.62 930278 1163094  10 172.94 1298434 1096393  11 238.57 1803403 1071971  12 304.02 2532623 1147835 Low1 8.83 61255 1036897 Low2 10.52 78951 1131502 Low3 11.77 58654 754843 Med1 112.78 786435 1046058 Med2 105.19 837224 1197875 Med3 97.48 488198 756299 High1 203.97 1418622 1001608 High2 218.97 1603009 1047207 High3 215.75 797944 529832

2^(nd) Set of Data on Norendoxifen

Norendoxifen Re- Cal- ISTD sponse culated FileName Response Response Ratio Conc 19066 394909 1631694 0.040 8.564 19142 259927 1499594 0.029 −2.102 19157 236174 1567347 0.025 −5.625 19175 303474 1650879 0.031 −0.471 19183 286322 1485735 0.032 0.910 19198 285421 1509741 0.032 0.341 19208 276540 1620554 0.028 −2.520 19278 296552 1357729 0.036 4.901 19306 266001 1273966 0.035 3.408 19309 174801 1139771 0.026 −5.207 19324 128810 640489 0.034 2.215 19360 86290 458714 0.031 0.195 19384 78299 423790 0.031 −0.326 19393 95749 526445 0.030 −0.774 19397 67733 519145 0.022 −8.771 19419 101553 680002 0.025 −5.833 19443 62792 438535 0.024 −6.791 19461 52923 345089 0.026 −5.208 19500 49566 298820 0.028 −3.262 19513 52689 327418 0.027 −4.032 19539 54812 286635 0.032 0.679 20081 52468 350067 0.025 −5.750 20124 78695 417502 0.031 0.254 20140 73246 371366 0.033 1.612 20160 26072 312569 0.014 −16.105 20175 40725 344100 0.020 −10.658 20188 70099 395249 0.030 −1.477 20205 133509 799351 0.028 −3.083 21436 105573 614214 0.029 −2.327 21546 94366 576559 0.027 −3.604 21564 59146 471390 0.021 −9.549 21603 54821 319202 0.029 −2.349 21630 60510 396701 0.025 −5.337 21640 56132 341040 0.027 −3.461 21653 47204 317333 0.025 −5.925 23320 40993 290890 0.023 −7.143 23339 20491 251970 0.014 −16.431 23359 28032 200226 0.023 −7.287 23390 12227 296939 0.007 −22.698 23413 211515 1259888 0.028 −2.949 23435 53419 263667 0.034 2.446 23438 25072 119561 0.035 3.548 25961 38335 315946 0.020 −10.194 26081 48157 246034 0.033 1.379 26155 52963 746052 0.012 −18.043 26206 26548 187122 0.024 −6.995 26226 43686 258528 0.028 −2.778 27064 48415 195754 0.041 9.386 27106 142236 953374 0.025 −5.857 27135 64096 643672 0.017 −13.584 27186 15662 159454 0.016 −13.796 27224 28810 253163 0.019 −11.367 27229 24116 193237 0.021 −9.654 QC_High_1_150506114043 2019214 1405526 0.239 189.933 QC_Low_1_150506112054 346367 1391580 0.041 9.630 QC_Med_1_150506113049 1144208 1372839 0.139 99.309 QC_High_2 295558 291585 0.169 126.576 QC_High_3 616664 396020 0.260 207.821 QC_Low_2 103046 372310 0.046 13.949 QC_Low_3 59735 259337 0.038 6.751 QC_Med_2 325974 367929 0.148 107.274 QC_Med_3 234615 392400 0.100 63.396 Std_1_150506091206 295660 1439901 0.034 2.870 Std_2_150506092201 256587 1378734 0.031 −0.117 Std_3_150506093155 285577 1349969 0.035 3.834 Std_4_150506094149 415667 1443768 0.048 15.673 Std_5_150506095143 317257 1315001 0.040 8.445 Std_6_150506100138 477087 1361340 0.058 25.345 Std_7_150506101134 843486 1415920 0.099 63.062 Std_8_150506102128 1055968 1323738 0.133 93.879 Std_9_150506103122 1310733 1333882 0.164 121.899 Std_10_150506104117 1861199 1339576 0.232 182.904 Std_11_150506105111 2398059 1352704 0.295 239.644 Std_12_150506110106 2913618 1341519 0.362 297.950

Endoxifen Quantitation and Response in Patient Samples.

Endoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.39 858 948878 18788 1.70 15846 764437 18824 2.67 28234 793359 18837 4.78 43744 645545 18865 6.58 74592 784027 18873 6.51 80112 851690 18885 5.08 103654 1434599 20434 NA NF 1524835 20478 NA NF 1873595 20484 1.30 20727 1411744 20511 1.66 29587 1466708 20515 2.65 49078 1392281 20564 3.22 65950 1498317 20573 5.34 111280 1461115 20581 3.80 75349 1426339 20601 7.58 176533 1599850 20647 14.34 317868 1490262 20661 13.54 284666 1415658 20689 12.57 267783 1436705 20704 15.68 340779 1458681 26619 0.34 114 1508141 26690 0.34 134 1475191 26756 1.90 34462 1447066 26777 3.36 59301 1287689 26801 5.40 102120 1323903 26826 6.68 92705 959275 26837 0.36 514 1115449 26853 7.38 101415 944719 26864 7.57 90589 822666 26878 10.16 118857 794035 26936 2.46 26351 812995 26952 3.78 46504 886924 26958 0.34 105 744284 26978 4.26 53111 887541 27009 5.99 71343 828349 27011 0.66 4334 876309 27022 6.65 66263 689142 27045 1.26 9665 686202 27067 0.94 5715 615842 27080 1.36 12324 785747 27084 1.42 13202 798627 27413 NA NF 465635 27446 1.29 9211 630981 27482 2.88 24964 643410 27526 7.75 68442 606397 27539 5.66 46555 574207  3002 0.35 81 466484 30031 1.10 5772 495349 30043 2.54 16964 505290 30079 4.20 29712 505353 30105 4.77 50235 743460 30116 5.34 55768 731395 30127 6.71 61146 629546  6420 0.41 1270 1070269  6482 2.62 39100 1121187  6497 4.72 77527 1162006  6510 4.39 47391 767435  6521 3.00 27958 687725  6535 7.94 102261 882954  6549 10.80 155921 978851 Std_1 1.13 12801 1062595 Std_2 2.69 38395 1069320 Std_3 4.75 76211 1132746 Std_4 14.64 230116 1056612 Std_5 12.13 210594 1172890 Std_6 23.81 416910 1166491 Std_7 59.39 1030203 1145572 Std_8 94.50 1638868 1143017 Std_9 117.78 2079955 1163094 Std_10 177.17 2952223 1096393 Std_11 248.84 4056331 1071971 Std_12 298.59 5213065 1147835 LowQC_1 10.20 155750 1036897 LowQC_2 10.70 178553 1131502 LowQC_3 8.24 90814 754843 MedQC_1 93.72 1487561 1046058 MedQC_2 87.15 1583496 1197875 MedQC_3 83.55 958371 756299 HighQC_1 200.24 3048922 1001608 HighQC_2 200.35 3189428 1047207 HighQC_3 206.53 1663579 529832

4-Hydroxy Tamoxifen Quantitation and Response in Patient Samples.

4-Hydroxy Tamoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.19 3015 2399530 18788 0.82 39658 2014612 18824 0.86 48913 2359977 18837 1.40 65306 1789387 18865 1.47 81483 2110634 18873 1.56 89856 2177221 18885 0.85 85218 4167738 20434 0.26 14678 4442630 20478 0.18 5170 5585352 20484 0.79 74921 3980217 20511 0.82 80728 4131271 20515 1.10 112006 4017151 20564 1.01 108651 4297841 20573 1.26 139523 4297129 20581 0.98 98939 4068467 20601 1.33 153875 4442435 20647 2.16 240633 4086174 20661 2.06 232708 4153726 20689 1.68 185318 4128291 20704 1.97 222092 4174587 26619 0.20 6064 4244184 26690 0.19 5282 4016987 26756 0.67 60702 3942716 26777 0.94 79670 3447066 26801 1.08 98947 3623797 26826 1.01 67829 2695516 26837 0.22 6220 3050204 26853 1.14 73111 2526194 26864 0.94 45419 1965544 26878 0.98 55858 2298573 26936 0.86 48390 2314343 26952 0.65 36142 2454817 26958 0.16 946 2226312 26978 0.53 27434 2440319 27009 0.88 52076 2421466 27011 0.44 19970 2351863 27022 1.02 52595 2073596 27045 0.55 23486 2023385 27067 0.58 22972 1835775 27080 0.57 23575 1895425 27084 0.67 29589 1927373 27413 0.20 2281 1631839 27446 0.76 30067 1668786 27482 1.14 51599 1775070 27526 1.85 86472 1741700 27539 1.30 54185 1611552  3002 NA NF 1366983 30031 0.45 13164 1510509 30043 0.69 24596 1549883 30079 0.71 23917 1457036 30105 0.70 34945 2179008 30116 0.75 36168 2049184 30127 0.85 40172 1967785  6420 0.21 5225 2882321  6482 0.65 47148 3221437  6497 0.87 63944 3016193  6510 0.52 23933 2193645  6521 0.51 21842 2078718  6535 1.02 58001 2272084  6549 1.10 78339 2829487 Std_1 0.86 63333 3061651 Std_2 1.60 126439 2979412 Std_3 3.09 273498 3183743 Std_4 10.22 857888 2914286 Std_5 8.14 781525 3347055 Std_6 14.99 1460920 3367273 Std_7 37.62 3585531 3271921 Std_8 67.06 6043897 3087672 Std_9 79.79 7391602 3171813 Std_10 121.24 11120485 3136830 Std_11 162.00 14892020 3141049 Std_12 197.45 18470067 3194201 LowQC_1 2.60 198332 2764071 LowQC_2 2.40 215841 3282140 LowQC_3 2.46 124759 1845395 MedQC_1 42.35 3760367 3046944 MedQC_2 45.77 4055530 3039643 MedQC_3 44.83 2330598 1783433 HighQC_1 98.45 7900913 2746235 HighQC_2 95.55 8331858 2984061 HighQC_3 96.85 4580837 1618676

N-Desmethyl-4′-Hydroxy Quantitation and Response in Patient Samples.

N-Desmethyl-4′-Hydroxy Tam Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.11 6478 1418949 18788 0.21 11255 1246580 18824 0.35 20911 1409614 18837 0.65 31944 1156863 18865 1.18 71666 1432048 18873 1.09 71427 1542934 18885 1.21 135392 2638726 20434 0.10 11165 2633347 20478 0.11 16114 3369744 20484 0.22 24190 2554478 20511 0.21 21589 2429777 20515 0.51 54176 2518940 20564 0.83 92905 2629052 20573 0.38 43295 2681694 20581 0.02 2135 2579787 20601 0.71 83112 2763637 20647 1.70 195854 2715179 20661 1.34 154254 2699457 20689 1.45 157523 2552623 20704 1.93 218342 2659928 26619 0.11 12283 2716910 26690 0.05 4982 2542146 26756 0.17 16848 2385510 26777 0.19 16813 2133085 26801 0.41 32728 1894557 26826 0.03 2140 1727922 26837 0.06 4057 1662633 26853 0.47 30871 1544615 26864 0.40 22301 1322198 26878 0.57 35997 1484688 26936 0.24 13870 1392328 26952 0.31 19864 1512928 26958 0.06 3660 1460163 26978 0.35 23834 1593929 27009 0.54 35183 1530063 27011 0.16 10031 1519605 27022 0.01 465 1331431 27045 0.22 11683 1276177 27067 0.01 247 1070301 27080 0.44 21767 1168677 27084 0.58 29142 1185205 27413 NA NF 939826 27446 0.16 6985 1007163 27482 0.35 15820 1061662 27526 0.61 27483 1065272 27539 0.43 16534 912845  3002 0.00 24 728838 30031 0.03 1048 831337 30043 0.18 6591 887422 30079 0.04 1359 910191 30105 0.70 43026 1436724 30116 0.80 47225 1386344 30127 NA NF 1128675  6420 0.14 10059 1717733  6482 0.26 22674 2067584  6497 0.08 5571 1626375  6510 0.06 3541 1309114  6521 0.01 277 1436238  6535 0.03 1862 1366228  6549 0.77 59031 1799445 Std_1 1.19 96247 1904747 Std_2 2.26 190378 1981125 Std_3 4.86 413787 2001224 Std_4 15.95 1257807 1850417 Std_5 12.19 1091191 2100153 Std_6 23.52 2088988 2082546 Std_7 59.49 5210489 2048907 Std_8 99.12 8606656 2025612 Std_9 122.57 10690211 2031234 Std_10 172.83 15602263 2095230 Std_11 236.95 21323531 2079609 Std_12 305.13 26862139 2024971 LowQC_1 10.95 828830 1776536 LowQC_2 11.30 972714 2019974 LowQC_3 9.88 487998 1159721 MedQC_1 96.08 7621147 1850788 MedQC_2 98.24 8103790 1924467 MedQC_3 103.17 4505234 1018413 HighQC_1 219.32 16710705 1762852 HighQC_2 214.74 18007527 1940796 HighQC_3 210.16 10408228 1146566

4′-Hydroxy Tamoxifen Quantitation and Response in Patient Samples.

4′-Hydroxy Tamoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.24 3363 2172088 18788 1.19 28281 1916250 18824 1.81 48728 2080977 18837 2.83 62234 1655472 18865 3.33 93441 2096079 18873 2.95 85399 2172978 18885 2.59 133675 3894668 20434 0.18 2811 4045035 20478 0.19 3758 5065428 20484 1.06 48506 3768254 20511 0.95 44050 3859077 20515 1.90 93981 3806194 20564 2.61 135079 3902896 20573 2.17 104172 3669717 20581 2.65 133250 3788223 20601 2.46 140885 4336626 20647 4.14 210537 3761489 20661 3.71 186268 3728418 20689 3.44 180034 3899111 20704 3.68 191393 3871001 26619 0.20 3694 4050702 26690 0.24 5417 3832047 26756 0.80 34425 3712870 26777 1.19 48068 3249380 26801 1.83 66555 2809186 26826 1.78 54590 2375921 26837 0.20 2306 2467253 26853 1.86 54910 2272552 26864 1.79 44228 1918046 26878 1.60 42925 2094936 26936 1.27 34702 2185824 26952 1.31 39922 2433167 26958 NA NF 2339037 26978 1.22 32938 2163677 27009 1.79 52321 2255996 27011 0.76 18543 2137153 27022 1.68 45719 2117327 27045 1.36 30371 1779913 27067 2.09 48462 1770196 27080 1.73 38821 1737223 27084 1.89 39706 1622586 27413 0.26 2863 1567595 27446 0.78 14009 1553585 27482 1.30 27446 1686572 27526 2.44 48500 1505217 27539 1.98 34937 1353173  3002 0.23 1581 1130425 30031 0.39 4408 1249506 30043 1.13 20090 1446682 30079 1.58 24236 1201364 30105 1.32 35388 2136332 30116 0.81 18620 1974717 30127 1.31 30533 1863767  6420 0.17 1208 2562866  6482 0.86 30040 2947532  6497 1.13 35827 2567403  6510 0.62 12846 1902106  6521 0.66 15265 2072439  6535 1.74 46483 2067649  6549 2.09 71871 2626700 Std_1 0.83 26072 2692512 Std_2 1.63 57494 2747701 Std_3 3.15 120736 2866766 Std_4 10.27 378179 2673535 Std_5 8.41 344843 2987180 Std_6 14.49 607841 3035767 Std_7 36.08 1504777 3007474 Std_8 66.95 2652805 2860540 Std_9 81.81 3229939 2852941 Std_10 122.36 4801896 2844972 Std_11 159.35 6128640 2797026 Std_12 198.29 7827883 2880957 LowQC_1 2.13 73604 2637536 LowQC_2 2.20 79203 2747045 LowQC_3 2.20 57019 1978114 MedQC_1 37.03 1384891 2696422 MedQC_2 39.46 1535649 2805856 MedQC_3 37.65 8374841 603915 HighQC_1 98.68 3324791 2437845 HighQC_2 98.97 3758231 2747447 HighQC_3 99.99 2320954 1679577

2^(nd) Set of Data on 4′-Hydroxy Tamoxifen

4′-Hydroxy Tamoxifen Cal- Re- culated ISTD sponse FileName Conc Response Response Ratio 19066 0.303 22269 4453051 0.001 19142 1.579 94126 4123038 0.004 19157 1.838 112491 4252834 0.004 19175 3.419 215656 4439146 0.008 19183 1.895 110049 4037559 0.005 19198 2.053 121419 4120446 0.005 19208 1.938 119044 4274660 0.005 19278 6.753 391621 4107533 0.016 19306 6.685 283075 2998975 0.016 19309 1.328 51548 2667301 0.003 19324 5.933 159394 1901442 0.014 19360 6.746 108720 1141453 0.016 19384 3.972 74081 1315036 0.009 19393 5.312 91931 1223801 0.013 19397 1.563 20063 887271 0.004 19419 2.243 79687 2480872 0.005 19443 1.866 33589 1251374 0.004 19461 2.490 35518 998353 0.006 19500 2.593 28876 779966 0.006 19513 2.550 36568 1004244 0.006 19539 1.572 19542 859341 0.004 20081 0.586 8058 901020 0.001 20124 1.781 31939 1244689 0.004 20140 1.899 30547 1118830 0.005 20160 1.828 24689 937980 0.004 20175 1.554 19421 863716 0.004 20188 1.898 32090 1175536 0.005 20205 2.902 85174 2060234 0.007 21436 0.117 4844 2021993 0.000 21546 1.348 28821 1470305 0.003 21564 2.889 59191 1437790 0.007 21603 2.569 36899 1006018 0.006 21630 4.485 58183 915834 0.011 21640 4.175 61789 1044008 0.010 21653 2.931 31548 755613 0.007 23320 0.548 5803 689039 0.001 23339 1.272 19300 1041074 0.003 23359 2.257 21560 667047 0.005 23390 1.908 12775 465563 0.005 23413 2.272 151070 4644733 0.005 23435 2.232 19746 617606 0.005 23438 1.798 12771 493301 0.004 25961 0.253 4401 1024904 0.001 26081 −0.023 275 622530 0.000 26155 0.064 1040 630132 0.000 26206 0.544 4554 544708 0.001 26226 0.210 3536 955368 0.001 27064 0.026 629 557421 0.000 27106 1.523 21251 964060 0.004 27135 1.867 35016 1303640 0.004 27186 2.309 153437 4642861 0.006 27224 2.862 35270 864675 0.007 27229 2.767 17985 455820 0.007 QC_High_1_150506114043 83.229 4597542 3825262 0.200 QC_Low_1_150506112054 2.664 140705 3701906 0.006 QC_Med_1_150506113049 37.743 1894768 3535619 0.089 QC_High_2 88.423 1031519 806248 0.213 QC_High_3 97.226 1330667 942761 0.235 QC_Low_2 5.388 77980 1023477 0.013 QC_Low_3 3.554 30997 613995 0.008 QC_Med_2 36.281 601572 1168351 0.086 QC_Med_3 38.465 558288 1021937 0.091 Std_1_150506091206 0.731 41595 3790363 0.002 Std_2_150506092201 1.540 83002 3723933 0.004 Std_3_150506093155 3.449 170169 3472544 0.008 Std_4_150506094149 9.368 510688 3865765 0.022 Std_5_150506095143 8.242 408166 3510555 0.019 Std_6_150506100138 17.558 884480 3570164 0.041 Std_7_150506101134 38.848 2115043 3832867 0.092 Std_8_150506102128 65.521 3179778 3383142 0.157 Std_9_150506103122 81.313 4019062 3425228 0.196 Std_10_150506104117 116.630 5995135 3515037 0.284 Std_11_150506105111 156.899 8071959 3465385 0.388 Std_12_150506110106 203.503 10317099 3356583 0.512

N-Desmethyl Tamoxifen Quantitation and Response in Patient Samples.

N-Desmethyl Tamoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.45 9391 7013663 18788 4.72 997637 5658276 18824 6.28 1289400 5370746 18837 10.69 2081686 4945768 18865 13.75 3224539 5904185 18873 16.85 3287990 4885497 18885 10.42 6162583 15037937 20434 0.44 13754 14973184 20478 0.43 11994 19567366 20484 3.20 1640706 14365887 20511 3.53 1826641 14327269 20515 6.01 3287099 14345893 20564 7.67 4371978 14704733 20573 7.68 4103602 13787594 20581 7.93 4120139 13386871 20601 9.88 5544877 14310077 20647 17.73 9200162 12971400 20661 16.29 9061736 13940923 20689 14.58 7719066 13305153 20704 16.47 8717935 13260540 26619 0.42 2117 11009897 26690 0.41 476 13027769 26756 2.22 863574 11677656 26777 3.32 1080841 9084409 26801 5.08 1508009 7900022 26826 5.31 1381666 6897386 26837 0.43 3833 6334347 26853 5.69 1098479 5081545 26864 6.05 1179428 5108144 26878 6.60 1541585 6084624 26936 3.81 836363 6017575 26952 3.48 913220 7282434 26958 0.43 3622 5877387 26978 4.29 1042266 6573581 27009 6.17 1529707 6486246 27011 2.03 380969 5767996 27022 5.78 1236685 5625116 27045 3.84 739878 5267957 27067 4.82 1030933 5713837 27080 5.66 1169780 5447403 27084 7.11 1364187 4971917 27413 0.42 1108 4253160 27446 1.98 324056 5043427 27482 3.77 601493 4375000 27526 8.09 1379832 4387718 27539 6.35 1054868 4342974  3002 0.42 646 3952111 30031 1.91 235647 3844766 30043 3.87 551445 3901710 30079 5.32 748541 3728783 30105 6.47 1529920 6170754 30116 7.07 1516184 5559182 30127 8.18 1836861 5779548  6420 0.60 65522 8832583  6482 4.04 1193657 8036794  6497 6.87 2044396 7727684  6510 5.49 1336093 6433864  6521 3.69 886207 6614396  6535 9.40 2555509 6943069  6549 11.01 3043467 7012004 Std_1 5.31 2327253 11617390 Std_2 9.97 4708014 12033677 Std_3 19.38 937598 112070965 Std_4 61.49 27754533 11094301 Std_5 49.86 25321926 12503328 Std_6 100.46 49441618 12062805 Std_7 237.72 122201199 12562079 Std_8 399.25 210842807 12886373 Std_9 527.12 256541351 11865614 Std_10 744.46 358614050 11729628 Std_11 986.35 456157172 11246764 Std_12 1256.13 543500909 10507976 LowQC_1 41.15 17191778 10304237 LowQC_2 40.50 15299115 9318490 LowQC_3 41.88 8361785 4924263 MedQC_1 184.96 82256696 10876149 MedQC_2 176.73 62369080 8631542 MedQC_3 209.89 42232054 4918806 HighQC_1 750.34 301780826 9793035 HighQC_2 724.44 245346920 8247495 HighQC_3 735.41 155798565 5158860

2^(nd) Set Data on N-Desmethyl Tamoxifen

N-Desmethyl Tamoxifen Calculated Response FileName Conc Response ISTD Response Ratio 19066 2.065 1893566 17801219 0.018 19142 5.193 3804551 16024676 0.040 19157 5.814 4318122 16392626 0.044 19175 15.766 10576421 15542009 0.113 19183 6.688 4269419 14229706 0.050 19198 8.157 5383057 14886974 0.060 19208 5.735 3583739 13778023 0.043 19278 23.120 15673119 15850368 0.165 19306 27.789 11650590 9834496 0.197 19309 6.095 1976544 7182395 0.046 19324 19.251 3793229 4588877 0.138 19360 24.238 4034751 3895645 0.173 19384 19.202 3353221 4066704 0.137 19393 17.748 3052769 3998016 0.127 19397 4.674 678296 3144886 0.036 19419 5.292 846685 3505013 0.040 19443 5.792 838732 3195069 0.044 19461 7.933 1017100 2887748 0.059 19500 9.755 1273539 2971638 0.071 19513 8.469 1110230 2963301 0.062 19539 9.135 1132214 2812449 0.067 20081 2.233 330630 2915326 0.019 20124 5.001 720772 3142098 0.038 20140 5.948 944581 3510755 0.045 20160 7.198 913531 2842164 0.054 20175 7.559 1028737 3056595 0.056 20188 8.153 1243431 3440186 0.060 20205 8.805 2379243 6120056 0.065 21436 −0.161 82557 6275244 0.002 21546 5.755 1093811 4191694 0.043 21564 11.340 2276511 4599180 0.082 21603 8.428 1177190 3156499 0.062 21630 13.598 1742557 2955369 0.098 21640 11.957 1582178 3037601 0.087 21653 11.957 1155197 2217912 0.087 23320 2.693 317766 2394849 0.022 23339 3.836 487883 2701690 0.030 23359 6.647 782831 2623922 0.050 23390 5.517 428388 1706743 0.042 23413 7.243 5505399 17027960 0.054 23435 4.842 378474 1699292 0.037 23438 4.856 245223 1098217 0.037 25961 −0.352 17773 3444979 0.001 26081 −0.428 4928 2536463 0.000 26155 −0.435 3436 2053149 0.000 26206 −0.421 4116 1814687 0.000 26226 −0.300 21248 2901877 0.001 27064 −0.439 2871 1935606 0.000 27106 4.249 1742409 8806092 0.033 27135 4.598 555198 2612748 0.035 27186 6.489 517740 1774796 0.049 27224 9.361 728755 1768581 0.069 27229 7.278 668537 2058499 0.054 QC_High_1_150506114043 660.452 435185890 15447019 4.695 QC_Low_1_150506112054 41.893 28271647 15914440 0.296 QC_Med_1_150506113049 176.768 110777325 14852088 1.243 QC_High_2 666.689 82150225 2888204 4.741 QC_High_3 750.310 97111711 3027264 5.347 QC_Low_2 59.321 7953162 3170574 0.418 QC_Low_3 42.049 3274694 1836588 0.297 QC_Med_2 188.111 27973649 3523808 1.323 QC_Med_3 207.865 23539310 2682660 1.462 Std_1_150506091206 4.715 3382129 15558800 0.036 Std_2_150506092201 10.198 6944239 15530643 0.075 Std_3_150506093155 20.520 13113426 14905147 0.147 Std_4_150506094149 61.006 41603966 16130492 0.430 Std_5_150506095143 52.335 33708040 15218576 0.369 Std_6_150506100138 100.981 64831809 15215929 0.710 Std_7_150506101134 239.435 164606137 16277076 1.685 Std_8_150506102128 400.008 254165419 14991752 2.826 Std_9_150506103122 512.940 315263074 14461814 3.633 Std_10_150506104117 751.782 473125498 14719297 5.357 Std_11_150506105111 989.021 602805977 14168882 7.091 Std_12_150506110106 1254.549 755234278 13899320 9.056

Tamoxifen Quantitation and Response in Patient Samples.

Tamoxifen Calculated ISTD Sample ID Conc (ng/mL) Response Area 18768 0.19 11350 8657179 18788 64.52 2071277 6179811 18824 75.45 2323692 5927027 18837 118.82 3493130 5650329 18865 134.03 4231612 6064968 18873 125.15 4035948 6196900 18885 54.09 5012052 17841365 20434 0.25 29490 17918878 20478 0.05 15176 24669280 20484 39.16 3465206 17038973 20511 51.28 4521960 16978931 20515 58.91 5194907 16978612 20564 62.95 5586963 17086731 20573 71.94 6062564 16221260 20581 58.71 4699760 15412475 20601 78.44 6699955 16436717 20647 137.89 10249638 14278153 20661 106.10 9245452 16755833 20689 90.43 7455218 15860377 20704 116.51 9830326 16218100 26619 0.07 10074 14455436 26690 0.18 20386 15879616 26756 34.95 2383129 13131665 26777 44.56 2534522 10951927 26801 63.56 2682884 8125969 26826 48.06 1933458 7746196 26837 0.29 12406 6671459 26853 54.28 1547378 5488947 26864 55.06 1556148 5441919 26878 41.41 1545348 7185485 26936 40.27 1394728 6669012 26952 27.71 1107256 7694108 26958 0.33 16221 7897622 26978 19.13 754743 7589060 27009 50.25 1927906 7387061 27011 36.56 1358366 7154478 27022 49.21 1655736 6478414 27045 48.06 1561893 6258393 27067 49.26 1517871 5933913 27080 48.76 1474533 5822771 27084 40.15 1262719 6055936 27413 0.25 8576 5300227 27446 26.64 793351 5733720 27482 38.93 1066943 5277298 27526 65.81 1659523 4854108 27539 66.06 1601415 4666695  3002 0.26 7653 4493100 30031 12.60 296745 4522346 30043 30.38 776495 4921844 30079 33.53 811130 4658023 30105 26.33 941949 6886006 30116 20.53 705407 6611936 30127 36.97 1165680 6071292  6420 0.61 36915 10553272  6482 18.90 991389 10091984  6497 33.76 1545743 8815779  6510 18.08 687863 7318004  6521 14.28 572749 7707224  6535 56.47 2235005 7619942  6549 60.89 2473508 7820955 Std_1 5.07 368746 13870144 Std_2 9.87 743436 14447818 Std_3 20.91 1577523 14518514 Std_4 61.61 4389679 13716703 Std_5 50.98 3886987 14681961 Std_6 98.50 7499571 14642835 Std_7 232.22 18185150 14991747 Std_8 401.17 31914385 15135753 Std_9 504.67 38342009 14399860 Std_10 761.87 53303371 13136206 Std_11 1028.75 70717814 12781973 Std_12 1221.75 91208237 13784726 LowQC_1 37.88 2612589 13281768 LowQC_2 37.79 2103909 10721385 LowQC_3 39.37 1299297 6355004 MedQC_1 195.63 12368756 12119500 MedQC_2 208.87 9917798 9097656 MedQC_3 192.21 6176283 6160378 HighQC_1 753.79 47482016 11830479 HighQC_2 783.21 37796034 9053718 HighQC_3 792.09 22732114 5382465

2^(nd) Set of Data on Tamoxifen

Tamoxifen Calculated Response FileName Conc Response ISTD Response Ratio 19066 3.701 535351 21932598 0.004 19142 46.038 4826863 18743658 0.043 19157 42.676 4468571 18698711 0.040 19175 70.899 7177323 18180212 0.066 19183 40.556 3795801 16700119 0.038 19198 50.601 4877479 17253561 0.047 19208 48.961 4130546 15094531 0.046 19278 112.000 10718988 17223765 0.104 19306 109.572 7039806 11562039 0.101 19309 12.404 588320 8141233 0.012 19324 116.499 3251398 5023090 0.108 19360 115.255 2918929 4558052 0.107 19384 69.683 1852847 4774541 0.065 19393 93.264 2389964 4609258 0.086 19397 58.819 1228203 3743936 0.055 19419 64.295 1435152 4005502 0.060 19443 65.809 1333605 3637172 0.061 19461 76.288 1478646 3482622 0.071 19500 76.305 1542766 3632836 0.071 19513 68.110 1283532 3383306 0.063 19539 45.479 803096 3156336 0.042 20081 32.732 624362 3390153 0.031 20124 48.263 1018603 3775475 0.045 20140 68.004 1470256 3881451 0.063 20160 64.933 1272861 3517926 0.060 20175 75.348 1428138 3405371 0.070 20188 89.929 2033491 4066576 0.083 20205 70.704 2541456 6455164 0.066 21436 1.476 85028 6980140 0.002 21546 48.585 1345694 4955204 0.045 21564 95.890 2607984 4892543 0.089 21603 63.763 1355947 3815715 0.059 21630 84.113 1611409 3444209 0.078 21640 82.497 1572414 3426332 0.076 21653 50.235 829879 2956721 0.047 23320 40.814 567630 2481819 0.038 23339 56.680 920885 2911936 0.053 23359 82.388 947509 2067360 0.076 23390 56.566 728329 2307661 0.053 23413 62.246 4437177 12788110 0.058 23435 39.294 493294 2238856 0.037 23438 38.475 344868 1597971 0.036 25961 0.037 17722 4142657 0.001 26081 −0.254 6716 2510216 0.000 26155 −0.439 4001 2413366 0.000 26206 0.050 11022 2534035 0.001 26226 −0.200 14415 4846489 0.000 27064 −0.023 9656 2445806 0.001 27106 42.931 2980808 12399985 0.040 27135 50.710 917164 3237516 0.047 27186 52.669 544080 1849951 0.049 27224 69.349 849916 2200587 0.064 27229 43.288 561047 2314994 0.040 QC_High_1_150506114043 665.297 75125747 19977315 0.627 QC_Low_1_150506112054 42.707 4685691 19592959 0.040 QC_Med_1_150506113049 191.494 19331542 18158006 0.177 QC_High_2 702.467 14501479 3646902 0.663 QC_High_3 693.744 14196581 3616342 0.654 QC_Low_2 56.305 1102329 3508672 0.052 QC_Low_3 44.334 546776 2203665 0.041 QC_Med_2 195.004 4267239 3935737 0.181 QC_Med_3 231.177 3406054 2647533 0.214 Std_1_150506091206 4.730 590077 19625944 0.005 Std_2_150506092201 10.057 1163202 19597467 0.010 Std_3_150506093155 20.691 2175560 18458380 0.020 Std_4_150506094149 57.908 6569960 20338991 0.054 Std_5_150506095143 52.677 5527146 18790321 0.049 Std_6_150506100138 104.641 10935888 18805047 0.097 Std_7_150506101134 240.132 26496831 19823070 0.223 Std_8_150506102128 403.963 42234460 18680230 0.377 Std_9_150506103122 521.039 53364854 18220106 0.488 Std_10_150506104117 745.879 78023680 18448605 0.705 Std_11_150506105111 957.878 98106705 17914330 0.913 Std_12_150506110106 1277.813 136400695 18438987 1.233

Patient Data Summary

Sample N-DM- N-DM-4′-OH- 4-OH- 4′-OH- ID # Tamoxifen Tamoxifen Tamoxifen Endoxifen Norendoxifen Tamoxifen Tamoxifen 19066 3.701 2.065 0.766 1.391 8.564 0.332 0.303 19142 46.038 5.193 0.916 4.316 −2.102 1.047 1.579 19157 42.676 5.814 0.831 5.240 −5.625 1.054 1.838 19175 70.899 15.766 2.934 26.761 −0.471 1.779 3.419 19183 40.556 6.688 1.480 6.161 0.910 1.378 1.895 19198 50.601 8.157 1.345 6.282 0.341 1.217 2.053 19208 48.961 5.735 0.691 6.009 −2.520 0.753 1.938 19278 112.000 23.120 4.595 39.457 4.901 2.840 6.753 19306 109.572 27.789 5.282 37.314 3.408 3.227 6.685 19309 12.404 6.095 1.611 3.442 −5.207 0.765 1.328 19324 116.499 19.251 4.073 35.628 2.215 2.209 5.933 19360 115.255 24.238 4.243 44.038 0.195 2.461 6.746 19384 69.683 19.202 3.648 37.655 −0.326 1.757 3.972 19393 93.264 17.748 2.821 35.239 −0.774 1.565 5.312 19397 58.819 4.674 0.193 5.816 −8.771 1.414 1.563 19419 64.295 5.292 0.194 7.615 −5.833 1.386 2.243 19443 65.809 5.792 0.251 8.806 −6.791 1.407 1.866 19461 76.288 7.933 0.467 12.942 −5.208 1.592 2.490 19500 76.305 9.755 0.569 15.373 −3.262 1.611 2.593 19513 68.110 8.469 0.692 16.571 −4.032 1.350 2.550 19539 45.479 9.135 0.760 17.872 0.679 1.142 1.572 20081 32.732 2.233 0.296 2.256 −5.750 0.447 0.586 20124 48.263 5.001 0.518 4.950 0.254 1.023 1.781 20140 68.004 5.948 0.494 7.486 1.612 0.996 1.899 20160 64.933 7.198 0.477 8.130 −16.105 0.868 1.828 20175 75.348 7.559 0.634 8.067 −10.658 0.645 1.554 20188 89.929 8.153 0.847 10.347 −1.477 1.275 1.898 20205 70.704 8.805 0.660 8.673 −3.083 0.855 2.902 21436 1.476 −0.161 −0.119 0.085 −2.327 0.075 0.117 21546 48.585 5.755 0.213 1.539 −3.604 0.371 1.348 21564 95.890 11.340 0.705 3.078 −9.549 0.658 2.889 21603 63.763 8.428 0.655 2.686 −2.349 0.546 2.569 21630 84.113 13.598 1.362 4.806 −5.337 0.615 4.485 21640 82.497 11.957 1.328 3.512 −3.461 0.600 4.175 21653 50.235 11.957 1.349 3.711 −5.925 0.688 2.931 23320 40.814 2.693 0.290 0.784 −7.143 0.646 0.548 23339 56.680 3.836 −0.075 3.599 −16.431 0.917 1.272 23359 82.388 6.647 0.278 5.589 −7.287 1.174 2.257 23390 56.566 5.517 0.254 4.420 −22.698 1.011 1.908 23413 62.246 7.243 0.584 6.938 −2.949 0.976 2.272 23435 39.294 4.842 0.390 4.922 2.446 0.571 2.232 23438 38.475 4.856 0.431 5.276 3.548 0.655 1.798 25961 0.037 −0.352 −0.108 0.072 −10.194 0.075 0.253 26081 −0.254 −0.428 −0.133 0.063 1.379 0.023 −0.023 26155 −0.439 −0.435 −0.163 NA −18.043 0.001 0.064 26206 0.050 −0.421 −0.083 0.015 −6.995 0.062 0.544 26226 −0.200 −0.300 −0.150 NA −2.778 0.054 0.210 27064 −0.023 −0.439 −0.122 0.013 9.386 0.056 0.026 27106 42.931 4.249 0.148 4.712 −5.857 1.267 1.523 27135 50.710 4.598 0.264 5.862 −13.584 0.958 1.867 27186 52.669 6.489 0.189 7.137 −13.796 0.874 2.309 27224 69.349 9.361 0.934 14.139 −11.367 1.390 2.862 27229 43.288 7.278 0.562 12.816 −9.654 1.055 2.767

Example 5 Additional Clinical Quantitation and Response Study

The standard operating protocol of Examples 1-3 were used to quantitate tamoxifen and its metabolites in patient samples and were correlated to tamoxifen response.

4′-Hydroxy Tamoxifen

4′-Hydroxy Tamoxifen Specified % Calculated ISTD Response Sample ID Conc Diff Conc Level Response Response Ratio RT Std_1 0.800 −19 0.650 1 18875 2072069 0.002 3.3169 Std_2 1.600 4 1.671 2 51081 2234410 0.004 3.3316 Std_3 3.200 21 3.866 3 113684 2168822 0.009 3.3309 Std_4 10.000 −11 8.924 4 271360 2253046 0.020 3.3313 Std_5 8.000 4 8.305 5 256942 2291669 0.019 3.3313 Std_6 16.000 0 15.936 6 476695 2221478 0.036 3.3310 Std_7 40.000 −3 38.805 7 1195727 2297958 0.087 3.3309 Std_8 64.000 4 66.462 8 1978710 2229656 0.148 3.3314 Std_9 80.000 6 84.720 9 2500818 2216656 0.188 3.3313 Std_10 120.000 −10 108.549 10 3532184 2452070 0.240 3.3313 Std_11 160.000 4 166.177 11 5013004 2292376 0.364 3.3309 Std_12 200.000 0 199.566 12 6005444 2297913 0.436 3.3310 QC_Low_1 2.000 26 2.528 1 Low 79280 2304249 0.006 3.3026 QC_Low_2 2.000 11 2.212 1 Low 77290 2564284 0.005 3.3597 QC_Low_3 2.000 6 2.110 1 Low 87323 3034648 0.005 3.3885 QC_Med_1 40.000 1 40.300 2 Med 1299131 2404628 0.090 3.3167 QC_Med_2 40.000 −5 38.185 2 Med 1228576 2399191 0.085 3.3596 QC_Med_3 40.000 5 42.171 2 Med 1549585 2741809 0.094 3.3888 QC_High_1 100.000 1 101.497 3 High 3219275 2387664 0.225 3.3173 QC_High_2 100.000 −7 92.799 3 High 3020319 2446947 0.206 3.3600 QC_High_3 100.000 1 100.902 3 High 3751385 2798463 0.223 3.3884 45301 NA NA 4.590 NA 155747 2505735 0.010 3.3310 45390 NA NA 8.253 NA 322666 2896059 0.019 3.3454 45466 NA NA 3.246 NA 127068 2883372 0.007 3.3314 45485 NA NA 0.591 NA 22294 2680691 0.001 3.3315 45569 NA NA 0.019 NA 1596 2641180 0.000 3.4171 45634 NA NA 3.033 NA 121975 2960336 0.007 3.3453 45717 NA NA 4.887 NA 196316 2967875 0.011 3.3457 45750 NA NA 2.940 NA 119665 2995645 0.007 3.3460 45752 NA NA 6.298 NA 255549 3001506 0.014 3.3457 45798 NA NA 2.022 NA 91673 3323059 0.005 3.3453 45800 NA NA 5.486 NA 225841 3043295 0.012 3.3453 45810 NA NA 3.438 NA 133315 2857926 0.008 3.3457 45825 NA NA 6.583 NA 260550 2928766 0.015 3.3453 45835 NA NA 3.962 NA 156772 2918803 0.009 3.3461 45867 NA NA 3.108 NA 123680 2929971 0.007 3.3457 45946 NA NA 5.147 NA 199901 2869775 0.012 3.3457 46037 NA NA 5.643 NA 230964 3026251 0.013 3.3453 46147 NA NA 8.247 NA 335804 3016126 0.019 3.3601 46153 NA NA 3.098 NA 119539 2841244 0.007 3.3458 46180 NA NA 3.884 NA 153272 2910985 0.009 3.3453 46213 NA NA 2.493 NA 109506 3227714 0.006 3.3603 46221 NA NA 3.541 NA 130860 2723957 0.008 3.3745 46283 NA NA 3.892 NA 138762 2629759 0.009 3.3601 46301 NA NA 5.251 NA 186491 2624655 0.012 3.3743 46428 NA NA 3.622 NA 132933 2705965 0.008 3.3746 46442 NA NA 2.922 NA 114861 2893174 0.007 3.3744 46453 NA NA 2.527 NA 99000 2879043 0.006 3.3601 46484 NA NA 3.743 NA 144129 2839205 0.008 3.3747 46518 NA NA 3.108 NA 113910 2698427 0.007 3.3747 46541 NA NA 4.711 NA 181415 2844378 0.011 3.3740 46548 NA NA 4.970 NA 190113 2826310 0.011 3.3740 46606 NA NA 2.090 NA 76649 2689489 0.005 3.3744 46667 NA NA 10.765 NA 461374 3177987 0.024 3.3740 46717 NA NA 7.253 NA 304387 3106739 0.016 3.3744 46731 NA NA 4.273 NA 182477 3152075 0.010 3.3744 46735 NA NA 2.919 NA 137705 3471886 0.007 3.3744 46749 NA NA 3.876 NA 174803 3326210 0.009 3.3884 46841 NA NA 3.195 NA 152289 3510357 0.007 3.3884 46859 NA NA 6.391 NA 308879 3575840 0.014 3.3888 46870 NA NA 3.938 NA 187297 3508553 0.009 3.3741 46896 NA NA 6.647 NA 310768 3459381 0.015 3.3744 46915 NA NA 0.314 NA 15189 3317417 0.001 3.3884 B1121 NA NA −0.011 NA 712 3604644 0.000 3.3889 B423 NA NA 7.737 NA 286757 2744489 0.017 3.3456 B599 NA NA 5.042 NA 193874 2841340 0.011 3.3457 B614 NA NA 3.958 NA 161177 3004219 0.009 3.3454 B651 NA NA 3.046 NA 114124 2758319 0.007 3.3740 B702 NA NA 3.499 NA 121390 2557001 0.008 3.3748 Specified % Calculated ISTD Response Sample ID Conc Diff Conc Level Response Response Ratio RT Std_1 0.800 5 0.840 1 35249 2958711 0.002 3.3003 Std_2 1.600 8 1.735 2 72861 2980562 0.004 3.3093 Std_3 3.200 −2 3.124 3 141591 3225973 0.007 3.3200 Std_4 10.000 −5 9.538 4 424861 3179029 0.022 3.3208 Std_5 8.000 −3 7.745 5 355442 3274330 0.018 3.3195 Std_6 16.000 −4 15.293 6 734203 3429193 0.036 3.3091 Std_7 40.000 −4 38.406 7 1779515 3315673 0.089 3.3088 Std_8 64.000 3 65.945 8 2961260 3219341 0.153 3.3187 Std_9 80.000 3 82.622 9 3780953 3284353 0.192 3.3205 Std_10 120.000 −2 117.460 10 5720657 3503326 0.272 3.3206 Std_11 160.000 2 163.231 11 7179921 3173352 0.377 3.3199 Std_12 200.000 −1 197.662 12 9562222 3497821 0.456 3.3209 OC_Low_2 2.000 27 2.535 1 Low 32917 923798 0.006 3.3201 OC_Low_2 2.000 −2 1.964 1 Low 64960 2349338 0.005 3.3408 QC_Low_1 2.000 3 2.061 1 Low 97498 3361269 0.005 3.3205 QC_Low_3 2.000 12 2.234 1 Low 28941 920883 0.005 3.3500 QC_Med_1 40.000 −4 38.547 2 Med 1884050 3497570 0.090 3.3195 QC_Med_2 40.000 −2 39.081 2 Med 566023 1036456 0.091 3.3303 QC_Med_2 40.000 −14 34.202 2 Med 182282 381263 0.080 3.3413 QC_Med_3 40.000 −7 37.337 2 Med 341288 654060 0.087 3.3406 QC_High_1 100.000 −5 94.884 3 High 4598348 3480962 0.220 3.3193 QC_High_2 100.000 −3 96.711 3 High 1251484 929592 0.224 3.3315 QC_High_2 100.000 −4 95.878 3 High 578257 433233 0.222 3.3402 QC_High_3 100.000 6 105.842 3 High 892150 605869 0.245 3.3395 46917 NA NA 6.294 NA 359107 4069111 0.015 3.3105 46951 NA NA 6.037 NA 358297 4232608 0.014 3.3187 46960 NA NA 4.332 NA 259114 4262102 0.010 3.3205 46990 NA NA 3.019 NA 186373 4393941 0.007 3.3299 47076 NA NA 4.647 NA 286503 4393808 0.011 3.3313 47148 NA NA 3.378 NA 203309 4284871 0.008 3.3213 47204 NA NA 4.003 NA 246164 4381015 0.009 3.3207 47223 NA NA 5.265 NA 329471 4461284 0.012 3.3197 47259 NA NA 4.429 NA 280965 4520722 0.010 3.3307 47262 NA NA 2.216 NA 146133 4688581 0.005 3.3298 47271 NA NA 3.533 NA 210174 4237018 0.008 3.3204 47285 NA NA 2.812 NA 167507 4239139 0.007 3.3209 47287 NA NA 4.284 NA 247990 4124588 0.010 3.3198 47294 NA NA 6.148 NA 304761 3535140 0.014 3.3299 47296 NA NA 6.503 NA 415945 4561599 0.015 3.3196 47362 NA NA 6.575 NA 217818 2362652 0.015 3.3205 47398 NA NA 1.552 NA 56103 2564963 0.004 3.3303 47408 NA NA 4.236 NA 140137 2356955 0.010 3.3298 47429 NA NA 2.437 NA 61808 1804049 0.006 3.3213 47478 NA NA 6.839 NA 146298 1525812 0.016 3.3309 47507 NA NA 5.091 NA 72042 1008788 0.012 3.3297 47512 NA NA 5.454 NA 74965 979958 0.013 3.3307 47547 NA NA 8.056 NA 116192 1029098 0.019 3.3290 47555 NA NA 3.667 NA 57301 1112989 0.009 3.3297 47583 NA NA 6.473 NA 86404 952010 0.015 3.3196 47586 NA NA 5.818 NA 76240 934334 0.014 3.3410 47626 NA NA 3.746 NA 45479 864793 0.009 3.3094 47629 NA NA 3.136 NA 29948 679734 0.007 3.3413 47633 NA NA 3.606 NA 26615 525691 0.008 3.3304 47674 NA NA 0.093 NA 847 585035 0.000 3.3612 47696 NA NA 3.824 NA 250106 4658812 0.009 3.3297 47715 NA NA 5.750 NA 261070 3237523 0.013 3.3313 B109 NA NA 1.524 NA 14768 687552 0.004 3.3488 B124 NA NA 8.718 NA 30895 252876 0.020 3.3412 B138 NA NA 3.821 NA 75737 1411978 0.009 3.3510 B151 NA NA 4.594 NA 57488 891738 0.011 3.3414 B174 NA NA 2.520 NA 26797 756341 0.006 3.3405 B209 NA NA 6.388 NA 48930 546310 0.015 3.3391 B211 NA NA 3.127 NA 38515 876857 0.007 3.3300 B218 NA NA 3.539 NA 36919 742987 0.008 3.3303 B249 NA NA 5.207 NA 21627 296085 0.012 3.3407 B348 NA NA 3.459 NA 73040 1503576 0.008 3.3409 B379 NA NA 6.245 NA 28271 322831 0.015 3.3412 B416 NA NA 3.862 NA 17684 326151 0.009 3.3410 B417 NA NA 4.720 NA 176904 2671010 0.011 3.3198 B50 NA NA 0.111 NA 667 393861 0.000 3.3521 B77 NA NA 4.677 NA 89665 1366217 0.011 3.3421 B793 NA NA 5.094 NA 21269 297647 0.012 3.3317 B875 NA NA 3.932 NA 27943 506274 0.009 3.3405 B96 NA NA 2.546 NA 22119 617916 0.006 3.3514

N-Desmethyl Tamoxifen

N-Desmethyl Tamoxifen Specified % Calculated ISTD Response Sample ID Conc Diff Conc Level Response Response Ratio RT Std_1 5.000 0 4.995 1 2214356 10007701 0.037 4.6555 Std_2 10.000 −1 9.920 2 4350445 10345933 0.070 4.6415 Std_3 20.000 1 20.214 3 8828632 10550159 0.139 4.6408 Std_4 62.500 −5 59.587 4 24912812 10256233 0.405 4.6413 Std_5 50.000 3 51.472 5 22282634 10606046 0.350 4.6412 Std_6 100.000 1 101.387 6 42220636 10250550 0.686 4.6409 Std_7 250.000 −3 241.804 7 101199824 10335393 1.632 4.6408 Std_8 400.000 5 419.552 8 164136702 9675655 2.827 4.6413 Std_9 500.000 2 508.595 9 207205488 10081408 3.426 4.6412 Std_10 750.000 −4 722.443 10 297647976 10206088 4.861 4.6412 Std_11 1000.000 0 995.302 11 411465548 10253258 6.688 4.6408 Std_12 1250.000 1 1262.236 12 512184551 10075081 8.473 4.6409 QC_Low_1 40.000 5 41.976 1 Low 18949835 11037046 0.286 4.6412 QC_Low_2 40.000 3 41.137 1 Low 18965589 11268913 0.281 4.6695 QC_Low_3 40.000 −1 39.797 1 Low 20961957 12869496 0.271 4.6841 QC_Med_1 200.000 0 200.142 2 Med 86969927 10724951 1.352 4.6410 QC_Med_2 200.000 −2 195.105 2 Med 82826081 10476833 1.318 4.6695 QC_Med_3 200.000 −3 194.728 2 Med 94847560 12020557 1.315 4.6843 QC_High_1 800.000 1 806.621 3 High 349559336 10739364 5.425 4.6272 QC_High_2 800.000 −4 766.892 3 High 321869979 10399085 5.159 4.6699 QC_High_3 800.000 −3 772.284 3 High 369290721 11848163 5.195 4.6839 45301 NA NA 26.273 NA 11841632 10945501 0.180 4.6552 45390 NA NA 39.710 NA 18654553 11477691 0.271 4.6553 45466 NA NA 14.131 NA 7051102 11934357 0.098 4.6556 45485 NA NA 2.104 NA 1235242 11839387 0.017 4.6558 45569 NA NA −0.438 NA 17606 11822707 0.000 4.6553 45634 NA NA 18.338 NA 9228298 12127381 0.127 4.6552 45717 NA NA 10.770 NA 5532916 12163713 0.076 4.6556 45750 NA NA 4.916 NA 2717384 12460010 0.036 4.6559 45752 NA NA 23.367 NA 12033795 12478509 0.161 4.6699 45798 NA NA 8.081 NA 4514702 13045004 0.058 4.6695 45800 NA NA 12.381 NA 6600043 12691932 0.087 4.6552 45810 NA NA 21.579 NA 10724905 12022578 0.149 4.6557 45825 NA NA 33.817 NA 17048836 12292179 0.231 4.6696 45835 NA NA 11.219 NA 5770806 12199963 0.079 4.6704 45867 NA NA 12.834 NA 6456515 11992891 0.090 4.6699 45946 NA NA 12.791 NA 6297921 11736675 0.089 4.6557 46037 NA NA 10.380 NA 5360503 12208515 0.073 4.6695 46147 NA NA 44.324 NA 21087917 11638790 0.302 4.6700 46153 NA NA 13.246 NA 6744190 12151619 0.093 4.6700 46180 NA NA 13.893 NA 7099200 12215198 0.097 4.6696 46213 NA NA 13.928 NA 7116545 12214797 0.097 4.6702 46221 NA NA 26.106 NA 11555669 10748057 0.179 4.6701 46283 NA NA 24.324 NA 10709139 10676283 0.167 4.6843 46301 NA NA 29.768 NA 14013373 11455981 0.204 4.6842 46428 NA NA 9.931 NA 4563811 10842547 0.070 4.6845 46442 NA NA 6.573 NA 3284257 11519780 0.048 4.6843 46453 NA NA 14.676 NA 7224761 11788779 0.102 4.6844 46484 NA NA 15.352 NA 6937804 10836949 0.107 4.6846 46518 NA NA 5.940 NA 2822979 10878008 0.043 4.6846 46541 NA NA 12.145 NA 5456620 10689596 0.085 4.6839 46548 NA NA 17.369 NA 7686125 10648745 0.120 4.6839 46606 NA NA 13.382 NA 6230069 11114932 0.093 4.6843 46667 NA NA 54.075 NA 26635133 12073060 0.368 4.6839 46717 NA NA 32.189 NA 15985951 12099953 0.220 4.6843 46731 NA NA 23.463 NA 11781079 12167379 0.161 4.6843 46735 NA NA 10.658 NA 6231222 13836352 0.075 4.6844 46749 NA NA 18.278 NA 10047672 13245981 0.126 4.6839 46841 NA NA 23.201 NA 12927666 13499605 0.160 4.6839 46859 NA NA 35.736 NA 20430737 13949932 0.244 4.6843 46870 NA NA 9.464 NA 5495150 13667994 0.067 4.6840 46896 NA NA 39.389 NA 22095261 13704056 0.269 4.6843 46915 NA NA 1.258 NA 945809 13492832 0.012 4.6839 B1121 NA NA −0.449 NA 14423 13610861 0.000 4.6845 B423 NA NA 33.052 NA 15825521 11670269 0.226 4.6556 B599 NA NA 15.587 NA 7789173 11988574 0.108 4.6556 B614 NA NA 14.595 NA 7250764 11894417 0.102 4.6553 B651 NA NA 17.086 NA 8096715 11398887 0.118 4.6839 B702 NA NA 20.529 NA 8668370 10203235 0.142 4.6847 Specified % Calculated ISTD Response Sample ID Conc Diff Conc Level Response Response Ratio RT Std_1 5.000 4 5.216 1 3035366 12894595 0.039 4.6451 Std_2 10.000 5 10.513 2 6039685 13277968 0.076 4.6539 Std_3 20.000 0 19.997 3 12196051 14385916 0.141 4.6542 Std_4 62.500 −6 58.667 4 34713350 14166401 0.408 4.6556 Std_5 50.000 −2 48.758 5 29854945 14637029 0.340 4.6434 Std_6 100.000 −3 96.677 6 60835111 15109059 0.671 4.6533 Std_7 250.000 −1 247.393 7 150417601 14628708 1.714 4.6430 Std_8 400.000 5 418.186 8 249322031 14341070 2.898 4.6422 Std_9 500.000 0 502.301 9 311783615 14926066 3.481 4.6444 Std_10 750.000 −2 736.267 10 481407377 15705917 5.109 4.6445 Std_11 1000.000 0 1004.915 11 593251935 14160635 6.982 4.6440 Std_12 1250.000 0 1248.606 12 768772647 14749010 8.687 4.6449 OC_Low_2 40.000 −11 35.438 1 Low 5014390 3370741 0.248 4.6642 OC_Low_2 40.000 −1 39.697 1 Low 8530728 5126252 0.277 4.6752 QC_Low_1 40.000 0 40.102 1 Low 25849688 15378255 0.280 4.6547 QC_Low_3 40.000 4 41.700 1 Low 5035346 2882014 0.291 4.6636 QC_Med_1 200.000 −6 187.687 2 Med 122092292 15647367 1.300 4.6539 QC_Med_2 200.000 −22 156.276 2 Med 19686199 3029159 1.083 4.6544 QC_Med_2 200.000 −6 187.708 2 Med 9278718 1189027 1.301 4.6760 QC_Med_3 200.000 −7 185.679 2 Med 14183336 1837368 1.287 4.6646 QC_High_1 800.000 −5 756.162 3 High 475031778 15088636 5.247 4.6434 QC_High_2 800.000 −16 674.720 3 High 70649330 2515953 4.680 4.6456 QC_High_2 800.000 −8 733.618 3 High 46860519 1534366 5.090 4.6643 QC_High_3 800.000 −6 751.226 3 High 64370878 2058125 5.213 4.6634 46917 NA NA 26.132 NA 18119897 16442596 0.184 4.6552 46951 NA NA 17.249 NA 12118193 16511132 0.122 4.6630 46960 NA NA 18.233 NA 13027133 16815396 0.129 4.6548 46990 NA NA 4.714 NA 3672603 17112987 0.036 4.6638 47076 NA NA 17.044 NA 12399477 17092640 0.121 4.6554 47148 NA NA 10.751 NA 7738928 16653008 0.077 4.6557 47204 NA NA 21.609 NA 15138607 16552674 0.152 4.6551 47223 NA NA 26.069 NA 18575235 16896149 0.183 4.6536 47259 NA NA 24.482 NA 17901150 17318645 0.172 4.6653 47262 NA NA 11.522 NA 8873081 17865097 0.083 4.6639 47271 NA NA 16.032 NA 11167382 16337683 0.114 4.6551 47285 NA NA 10.523 NA 6632132 14567325 0.076 4.6551 47287 NA NA 7.253 NA 4150522 12977664 0.053 4.6644 47294 NA NA 23.672 NA 10097368 10096633 0.167 4.6642 47296 NA NA 25.731 NA 18048131 16627998 0.181 4.6639 47362 NA NA 30.720 NA 9623565 7447965 0.215 4.6651 47398 NA NA 11.900 NA 2953091 5764235 0.085 4.6647 47408 NA NA 14.802 NA 2642033 4176803 0.105 4.6641 47429 NA NA 11.723 NA 2739959 5425673 0.084 4.6662 47478 NA NA 26.901 NA 5450199 4806728 0.189 4.6650 47507 NA NA 25.265 NA 3608824 3385136 0.178 4.6642 47512 NA NA 22.956 NA 2507451 2583917 0.162 4.6653 47547 NA NA 39.779 NA 5279817 3166205 0.278 4.6629 47555 NA NA 15.859 NA 2083193 3080093 0.113 4.6742 47583 NA NA 8.888 NA 929424 2398272 0.065 4.6641 47586 NA NA 30.921 NA 3210173 2468503 0.217 4.6650 47626 NA NA 18.322 NA 1864197 2394881 0.130 4.6640 47629 NA NA 5.135 NA 450948 1943285 0.039 4.6761 47633 NA NA 12.866 NA 967726 1751989 0.092 4.6645 47674 NA NA −0.454 NA 874 1612859 0.000 4.6644 47696 NA NA 16.209 NA 3169773 4588361 0.115 4.6641 47715 NA NA 13.470 NA 1656231 2868681 0.096 4.6758 B109 NA NA 11.432 NA 1033667 2097056 0.082 4.6825 B124 NA NA 14.692 NA 692056 1101989 0.105 4.6754 B138 NA NA 22.437 NA 1400162 1475589 0.158 4.6752 B151 NA NA 21.416 NA 1047332 1155265 0.151 4.6656 B174 NA NA 10.349 NA 412904 921568 0.075 4.6644 B209 NA NA 14.006 NA 798577 1331885 0.100 4.6731 B211 NA NA 11.346 NA 432864 884514 0.082 4.6748 B218 NA NA 14.915 NA 990100 1553714 0.106 4.6751 B249 NA NA 15.747 NA 589399 877488 0.112 4.6749 B348 NA NA 15.742 NA 1252157 1864674 0.112 4.6755 B379 NA NA 16.374 NA 789263 1131237 0.116 4.6754 B416 NA NA 13.889 NA 652860 1097759 0.099 4.6655 B417 NA NA 17.747 NA 4252133 5635185 0.126 4.6645 B50 NA NA −0.344 NA 10474 2062412 0.001 4.6659 B77 NA NA 13.213 NA 997904 1760839 0.094 4.6666 B793 NA NA 16.843 NA 766191 1068454 0.120 4.6764 B875 NA NA 19.567 NA 1696007 2043385 0.138 4.6748 B96 NA NA 9.888 NA 952563 2220513 0.071 4.6755

Tamoxifen

Tamoxifen Specified % Calculated ISTD Sample Response Sample ID Conc Diff Conc Level Response Response Type Ratio RT Std_1 5.000 −5 4.772 1 359548 11850488 Std Bracket 0.005 4.8747 Sample Std_2 10.000 0 10.034 2 715154 12408704 Std Bracket 0.010 4.8751 Sample Std_3 20.000 4 20.855 3 1400493 12310503 Std Bracket 0.019 4.8744 Sample Std_4 62.500 −6 58.586 4 3787383 12232259 Std Bracket 0.052 4.8748 Sample Std_5 50.000 8 53.761 5 3508560 12329561 Std Bracket 0.047 4.8748 Sample Std_6 100.000 −3 97.395 6 6307063 12335431 Std Bracket 0.085 4.8745 Sample Std_7 250.000 −2 245.182 7 15411820 12028313 Std Bracket 0.214 4.8744 Sample Std_8 400.000 4 417.365 8 25925506 11879210 Std Bracket 0.364 4.8749 Sample Std_9 500.000 3 514.348 9 31812253 11817737 Std Bracket 0.449 4.8748 Sample Std_10 750.000 −5 709.105 10 45628283 12268396 Std Bracket 0.620 4.8748 Sample Std_11 1000.000 1 1008.577 11 66892579 12598429 Std Bracket 0.885 4.8601 Sample Std_12 1250.000 1 1257.491 12 77548728 11676394 Std Bracket 1.107 4.8601 Sample QC_Low_1 40.000 1 40.559 1 Low 2820699 13057549 QC Sample 0.036 4.8604 QC_Low_2 40.000 −6 37.761 1 Low 2690867 13354380 QC Sample 0.034 4.9031 QC_Low_3 40.000 −11 35.695 1 Low 2960468 15518087 QC Sample 0.032 4.9177 QC_Med_1 200.000 3 205.744 2 Med 13931187 12953193 QC Sample 0.179 4.8602 QC_Med_2 200.000 −9 182.179 2 Med 12123620 12726226 QC Sample 0.159 4.9032 QC_Med_3 200.000 −6 187.142 2 Med 14410318 14726719 QC Sample 0.163 4.9035 QC_High_1 800.000 3 820.859 3 High 54350709 12607050 QC Sample 0.719 4.8607 QC_High_2 800.000 −9 729.436 3 High 48823606 12758558 QC Sample 0.638 4.9035 QC_High_3 800.000 −6 750.663 3 High 55810349 14168310 QC Sample 0.657 4.9032 45301 NA NA 130.875 NA 9201591 13424057 Unknown 0.114 4.8745 Sample 45390 NA NA 142.588 NA 10729349 14374421 Unknown 0.124 4.8889 Sample 45466 NA NA 51.861 NA 3887923 14153276 Unknown 0.046 4.8892 Sample 45485 NA NA 2.025 NA 225794 14029301 Unknown 0.003 4.8893 Sample 45569 NA NA −0.642 NA 31725 13993172 Unknown 0.000 4.9032 Sample 45634 NA NA 79.422 NA 6087679 14568254 Unknown 0.070 4.8888 Sample 45717 NA NA 114.657 NA 8769809 14590327 Unknown 0.100 4.8892 Sample 45750 NA NA 26.715 NA 2100131 14567323 Unknown 0.024 4.8895 Sample 45752 NA NA 103.765 NA 7994477 14684389 Unknown 0.091 4.8892 Sample 45798 NA NA 35.068 NA 2844990 15171845 Unknown 0.031 4.8888 Sample 45800 NA NA 50.405 NA 3867320 14476922 Unknown 0.045 4.8888 Sample 45810 NA NA 115.360 NA 8718130 14416627 Unknown 0.101 4.8893 Sample 45825 NA NA 179.654 NA 13173726 14022261 Unknown 0.157 4.8889 Sample 45835 NA NA 116.025 NA 8544018 14048402 Unknown 0.101 4.8896 Sample 45867 NA NA 90.584 NA 6772504 14231363 Unknown 0.079 4.8892 Sample 45946 NA NA 68.180 NA 5068384 14099973 Unknown 0.060 4.8893 Sample 46037 NA NA 122.733 NA 9267700 14411275 Unknown 0.107 4.8888 Sample 46147 NA NA 245.606 NA 18005272 14028174 Unknown 0.214 4.8892 Sample 46153 NA NA 64.050 NA 4709356 13932869 Unknown 0.056 4.8893 Sample 46180 NA NA 45.657 NA 3482346 14361051 Unknown 0.040 4.8888 Sample 46213 NA NA 39.700 NA 3002720 14193252 Unknown 0.035 4.8894 Sample 46221 NA NA 60.817 NA 4132298 12864807 Unknown 0.054 4.9037 Sample 46283 NA NA 106.601 NA 7203546 12882631 Unknown 0.093 4.9036 Sample 46301 NA NA 175.670 NA 12247590 13331077 Unknown 0.153 4.9035 Sample 46428 NA NA 102.971 NA 7021703 12996179 Unknown 0.090 4.9037 Sample 46442 NA NA 34.971 NA 2612135 13967304 Unknown 0.031 4.9036 Sample 46453 NA NA 76.528 NA 5324388 13217410 Unknown 0.067 4.9036 Sample 46484 NA NA 134.492 NA 9125489 12957248 Unknown 0.117 4.9038 Sample 46518 NA NA 32.697 NA 2256501 12878575 Unknown 0.029 4.9182 Sample 46541 NA NA 77.359 NA 4843270 11895464 Unknown 0.068 4.9175 Sample 46548 NA NA 85.387 NA 5732477 12770877 Unknown 0.075 4.9032 Sample 46606 NA NA 59.848 NA 4199528 13282068 Unknown 0.053 4.9179 Sample 46667 NA NA 135.598 NA 10414841 14668087 Unknown 0.118 4.9032 Sample 46717 NA NA 182.112 NA 14165239 14874821 Unknown 0.159 4.9036 Sample 46731 NA NA 145.655 NA 10828856 14203833 Unknown 0.127 4.9036 Sample 46735 NA NA 60.560 NA 5245978 16400049 Unknown 0.053 4.9180 Sample 46749 NA NA 97.135 NA 8098485 15881133 Unknown 0.085 4.9032 Sample 46841 NA NA 91.310 NA 7725533 16106190 Unknown 0.080 4.9032 Sample 46859 NA NA 129.621 NA 11198473 16494335 Unknown 0.113 4.9036 Sample 46870 NA NA 94.153 NA 7837005 15850306 Unknown 0.082 4.9176 Sample 46896 NA NA 152.754 NA 13132709 16429150 Unknown 0.133 4.9035 Sample 46915 NA NA 2.000 NA 257251 16113116 Unknown 0.003 4.9032 Sample B1121 NA NA −0.601 NA 39714 16006905 Unknown 0.000 4.9181 Sample B423 NA NA 140.264 NA 10248102 13955854 Unknown 0.122 4.8748 Sample B599 NA NA 68.107 NA 5070892 14121922 Unknown 0.060 4.8892 Sample B614 NA NA 74.326 NA 5545621 14169095 Unknown 0.065 4.8889 Sample B651 NA NA 63.727 NA 4449989 13231237 Unknown 0.056 4.9175 Sample B702 NA NA 67.581 NA 4315968 12111629 Unknown 0.059 4.9040 Sample

Specified Calculated % ISTD Response Sample ID Conc Conc Diff Level Response Response Ratio RT Std_1 5.000 5.166 3 1 518618 15827044 0.005 4.8732 Std_2 10.000 10.599 6 2 987999 15876927 0.010 4.8716 Std_3 20.000 19.658 −2 3 1961473 17615708 0.019 4.8825 Std_4 62.500 60.153 −4 4 5563829 16808158 0.055 4.8734 Std_5 50.000 48.987 −2 5 4721538 17459086 0.045 4.8716 Std_6 100.000 96.775 −3 6 9592859 18106874 0.088 4.8713 Std_7 250.000 244.797 −2 7 23979787 17971445 0.222 4.8711 Std_8 400.000 412.508 3 8 40252034 17905472 0.375 4.8703 Std_9 500.000 509.298 2 9 49965048 17996378 0.463 4.8726 Std_10 750.000 732.247 −2 10 78874481 19736612 0.666 4.8726 Std_11 1000.000 1014.448 1 11 96247175 17353278 0.924 4.8823 Std_12 1250.000 1242.858 −1 12 128307307 18853095 1.134 4.8731 OC_Low_2 40.000 41.992 5 1 Low 775940 3337584 0.039 4.8924 OC_Low_2 40.000 33.880 −15 1 Low 743748 3945978 0.031 4.8930 QC_Low_1 40.000 38.644 −3 1 Low 3934913 18359852 0.036 4.8829 QC_Low_3 40.000 42.899 7 1 Low 775947 3268415 0.040 4.8917 QC_Med_1 200.000 189.461 −5 2 Med 19360578 18735738 0.172 4.8820 QC_Med_2 200.000 186.560 −7 2 Med 3045361 2992744 0.170 4.8825 QC_Med_2 200.000 201.031 1 2 Med 1686040 1537992 0.183 4.8938 QC_Med_3 200.000 187.608 −6 2 Med 2096153 2048464 0.171 4.8928 QC_High_1 800.000 769.738 −4 3 High 78604327 18706868 0.700 4.8716 QC_High_2 800.000 705.358 −12 3 High 10431828 2710267 0.642 4.8840 QC_High_2 800.000 721.156 −10 3 High 5900132 1499181 0.656 4.8925 QC_High_3 800.000 819.576 2 3 High 10644697 2378535 0.746 4.8814 46917 NA 114.854 NA NA 12137244 19328077 0.105 4.8730 46951 NA 142.801 NA NA 14998177 19234280 0.130 4.8807 46960 NA 98.319 NA NA 10565017 19631305 0.090 4.8829 46990 NA 28.847 NA NA 3132543 19434679 0.027 4.8920 47076 NA 101.708 NA NA 11142969 20020581 0.093 4.8835 47148 NA 59.043 NA NA 6308156 19409932 0.054 4.8838 47204 NA 102.845 NA NA 10718957 19047492 0.094 4.8833 47223 NA 78.569 NA NA 8469906 19653465 0.072 4.8817 47259 NA 114.884 NA NA 12650349 20139926 0.105 4.8935 47262 NA 46.850 NA NA 5288079 20429903 0.043 4.8920 47271 NA 82.980 NA NA 8646221 19006812 0.076 4.8834 47285 NA 54.159 NA NA 4680113 15679142 0.050 4.8833 47287 NA 36.896 NA NA 2820080 13767116 0.034 4.8823 47294 NA 84.735 NA NA 5185004 11164296 0.077 4.8923 47296 NA 104.447 NA NA 10852415 18991199 0.095 4.8817 47362 NA 115.191 NA NA 4816356 7647588 0.105 4.8932 47398 NA 28.116 NA NA 905707 5760762 0.026 4.8929 47408 NA 102.226 NA NA 2846054 5087819 0.093 4.8923 47429 NA 56.922 NA NA 1821271 5809718 0.052 4.8843 47478 NA 143.152 NA NA 3596365 4600874 0.130 4.8931 47507 NA 131.205 NA NA 2452209 3421199 0.119 4.8923 47512 NA 85.263 NA NA 1482894 3173362 0.078 4.8935 47547 NA 221.439 NA NA 3947988 3270236 0.201 4.8910 47555 NA 72.270 NA NA 1217277 3067942 0.066 4.8920 47583 NA 42.391 NA NA 594900 2535249 0.039 4.8922 47586 NA 136.423 NA NA 2212374 2969176 0.124 4.8932 47626 NA 84.268 NA NA 1417562 3069017 0.077 4.8921 47629 NA 42.703 NA NA 531982 2250912 0.039 4.9043 47633 NA 80.594 NA NA 794839 1798467 0.074 4.8826 47674 NA −0.750 NA NA 1647 2363729 0.000 4.9231 47696 NA 119.835 NA NA 3332289 5087393 0.109 4.8923 47715 NA 132.525 NA NA 1832445 2531225 0.121 4.9040 B109 NA 24.561 NA NA 393995 2856321 0.023 4.9004 B124 NA 87.045 NA NA 431391 904445 0.079 4.9035 B138 NA 97.370 NA NA 877117 1645556 0.089 4.9034 B151 NA 94.329 NA NA 715445 1385149 0.086 4.8937 B174 NA 56.169 NA NA 511797 1654158 0.052 4.8925 B209 NA 152.886 NA NA 1473081 1765109 0.139 4.8909 B211 NA 52.935 NA NA 329416 1128693 0.049 4.8927 B218 NA 77.648 NA NA 1039019 2439216 0.071 4.8930 B249 NA 91.092 NA NA 597510 1197560 0.083 4.8929 B348 NA 65.394 NA NA 867110 2412290 0.060 4.8935 B379 NA 149.254 NA NA 984468 1208202 0.136 4.8933 B416 NA 137.946 NA NA 873145 1158959 0.126 4.8937 B417 NA 119.721 NA NA 3160264 4829324 0.109 4.8927 B50 NA −0.596 NA NA 3075 2010696 0.000 4.9146 B77 NA 65.086 NA NA 805769 2252079 0.060 4.8948 B793 NA 102.335 NA NA 726520 1297409 0.093 4.8942 B875 NA 81.439 NA NA 1183494 2650382 0.074 4.8927 B96 NA 60.352 NA NA 974518 2934438 0.055 4.8933

Patient Data Summary

4-OH- 4′-OH- N-DM- N-DM-4′-OH Sample ID# Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Endoxifen Norendoxifen 45301 130.875 1.778 4.590 26.273 3.852 19.943 5.074 45390 142.588 2.672 8.253 39.710 9.206 17.691 7.059 45466 51.861 0.965 3.246 14.131 3.310 15.559 4.693 45485 2.025 0.487 0.591 2.104 1.092 1.168 1.957 45569 −0.642 NA 0.019 −0.438 −0.222 NA 0.564 45634 79.422 0.873 3.033 18.338 2.705 14.921 3.811 45717 114.657 2.677 4.887 10.770 2.395 60.815 4.888 45750 26.715 0.612 2.940 4.916 2.489 5.976 3.368 45752 103.765 1.911 6.298 23.367 5.130 12.995 5.275 45798 35.068 0.740 2.022 8.081 1.968 3.777 2.611 45800 50.405 1.669 5.486 12.381 6.013 12.760 7.030 45810 115.360 1.866 3.438 21.579 3.861 32.991 4.740 45825 179.654 1.142 6.583 33.817 5.314 20.421 4.487 45835 116.025 2.009 3.962 11.219 1.776 29.102 3.337 45867 90.584 1.537 3.108 12.834 2.016 29.304 4.865 45946 68.180 1.175 5.147 12.791 3.843 16.281 5.364 46037 122.733 2.761 5.643 10.380 2.595 59.095 4.910 46147 245.606 1.436 8.247 44.324 5.977 24.870 4.687 46153 64.050 1.205 3.098 13.246 2.827 17.213 3.271 46180 45.657 0.924 3.884 13.893 4.240 6.037 3.156 46213 39.700 2.014 2.493 13.928 3.511 7.792 4.534 46221 60.817 1.115 3.541 26.106 5.312 16.512 5.436 46283 106.601 1.252 3.892 24.324 4.666 12.331 3.605 46301 175.670 1.076 5.251 29.768 5.016 9.789 2.748 46428 102.971 1.898 3.622 9.931 1.865 25.805 3.573 46442 34.971 0.620 2.922 6.573 2.995 6.342 3.827 46453 76.528 0.864 2.527 14.676 2.028 13.113 2.850 46484 134.492 1.441 3.743 15.352 2.667 30.740 4.162 46518 32.697 0.721 3.108 5.940 3.264 8.837 6.699 46541 77.359 1.187 4.711 12.145 3.176 17.268 3.617 46548 85.387 1.205 4.970 17.369 5.486 7.076 3.513 46606 59.848 0.842 2.090 13.382 2.749 9.943 3.175 46667 135.598 5.743 10.765 54.075 16.979 23.458 12.284 46717 182.112 1.902 7.253 32.189 5.665 22.140 5.021 46731 145.655 0.965 4.273 23.463 5.254 9.207 3.454 46735 60.560 1.081 2.919 10.658 2.891 17.339 4.412 46749 97.135 1.764 3.876 18.278 3.681 25.845 4.007 46841 91.310 1.679 3.195 23.201 3.203 43.515 4.586 46859 129.621 3.643 6.391 35.736 7.708 21.258 8.215 46870 94.153 2.574 3.938 9.464 2.031 59.243 5.910 46896 152.754 1.602 6.647 39.389 7.929 9.876 5.884 46915 2.000 0.189 0.314 1.258 0.712 2.050 3.135 B1121 −0.601 0.014 −0.011 −0.449 −0.140 0.264 0.617 B423 140.264 2.679 7.737 33.052 5.703 42.457 11.239 B599 68.107 0.976 5.042 15.587 4.554 6.927 2.742 B614 74.326 1.309 3.958 14.595 3.299 26.725 6.162 B651 63.727 0.571 3.046 17.086 4.085 9.597 5.964 B702 67.581 1.861 3.499 20.529 4.310 24.278 7.953 46917 114.854 1.781 6.294 26.132 5.430 10.963 3.918 46951 142.801 2.110 6.037 17.249 3.246 40.458 7.199 46960 98.319 0.564 4.332 18.233 3.288 9.167 4.594 46990 28.847 0.516 3.019 4.714 2.314 5.317 1.989 47076 101.708 1.806 4.647 17.044 2.692 28.357 4.324 47148 59.043 0.966 3.378 10.751 2.377 19.781 4.055 47204 102.845 0.580 4.003 21.609 3.480 11.477 6.512 47223 78.569 1.250 5.265 26.069 6.264 17.728 5.223 47259 114.884 0.793 4.429 24.482 4.635 14.089 3.709 47262 46.850 0.562 2.216 11.522 2.477 9.176 3.248 47271 82.980 1.230 3.533 16.032 3.110 20.053 3.197 47285 54.159 0.606 2.812 10.523 2.169 8.497 2.922 47287 36.896 0.913 4.284 7.253 2.942 11.271 5.726 47294 84.735 1.479 6.148 23.672 4.107 28.117 8.311 47296 104.447 1.532 6.503 25.731 5.416 11.705 5.398 47362 115.191 1.411 6.575 30.720 5.126 22.343 4.939 47398 28.116 0.550 1.552 11.900 1.966 10.628 3.597 47408 102.226 2.128 4.236 14.802 2.134 38.171 3.988 47429 56.922 0.848 2.437 11.723 2.483 20.228 7.582 47478 143.152 1.224 6.839 26.901 6.114 14.565 7.589 47507 131.205 0.642 5.091 25.265 4.416 7.048 3.479 47512 85.263 0.779 5.454 22.956 5.723 5.756 2.200 47547 221.439 1.777 8.056 39.779 6.325 22.285 5.877 47555 72.270 0.968 3.667 15.859 3.213 11.421 0.709 47583 42.391 1.163 6.473 8.888 3.863 10.629 7.439 47586 136.423 1.214 5.818 30.921 5.091 7.639 2.995 47626 84.268 0.511 3.746 18.322 3.172 8.915 9.121 47629 42.703 0.374 3.136 5.135 2.413 5.126 2.355 47633 80.594 0.784 3.606 12.866 1.604 23.162 6.992 47674 −0.750 −0.071 0.093 −0.454 −0.234 0.223 2.439 47696 119.835 1.648 3.824 16.209 2.188 32.499 4.597 47715 132.525 1.562 5.750 13.470 2.948 24.293 4.958 B109 24.561 0.040 1.524 11.432 2.366 1.434 2.485 B124 87.045 1.935 8.718 14.692 3.990 25.290 6.882 B138 97.370 1.675 3.821 22.437 2.896 37.164 4.369 B151 94.329 0.461 4.594 21.416 3.940 7.651 3.439 B174 56.169 1.006 2.520 10.349 1.803 9.252 3.782 B209 152.886 1.928 6.388 14.006 2.088 33.909 33.557 B211 52.935 0.557 3.127 11.346 2.399 11.457 0.686 B218 77.648 1.382 3.539 14.915 2.274 19.591 2.085 B249 91.092 0.929 5.207 15.747 2.646 18.802 1.890 B348 65.394 0.420 3.459 15.742 2.069 10.935 1.837 B379 149.254 1.016 6.245 16.374 3.163 32.589 6.254 B416 137.946 1.436 3.862 13.889 1.218 26.686 0.898 B417 119.721 1.334 4.720 17.747 2.352 18.884 0.363 B50 −0.596 −0.104 0.111 −0.344 −0.128 NA 0.088 B77 65.086 0.589 4.677 13.213 4.478 4.298 NA B793 102.335 1.034 5.094 16.843 3.572 17.569 1.393 B875 81.439 0.557 3.932 19.567 3.907 10.613 2.350 B96 60.352 0.437 2.546 9.888 2.496 14.510 15.071

The contents of the articles, patents, patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 

That which is claimed is:
 1. A method for determining an amount of norendoxifen in a human sample by mass spectrometry, comprising: (a) ionizing said norendoxifen to produce one or more norendoxifen ions detectable by mass spectrometry; (b) detecting the amount of the norendoxifen ion(s) from step (a) by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of norendoxifen in said sample, wherein the method has a limit of quantitation less than or equal to 5 ng/mL.
 2. The method of claim 1, further comprising protein precipitation prior to step (a).
 3. The method of claim 1, further comprising purification prior to step (a).
 4. The method of claim 3, wherein said purification comprises filtration.
 5. The method of claim 3, wherein said purification comprises liquid chromatography.
 6. The method of claim 5, wherein said liquid chromatography is high pressure liquid chromatography (HPLC).
 7. The method of claim 1, further comprising detecting the amount of an internal standard.
 8. The method of claim 7, wherein said internal standard is a deuterated norendoxifen.
 9. The method of claim 1, wherein said ionization is by atmospheric pressure chemical ionization (APCI).
 10. The method of claim 1, wherein said ionization is in positive ion mode.
 11. The method of claim 1, wherein said sample is a serum sample.
 12. The method of claim 1, wherein said mass spectrometry is tandem mass spectrometry.
 13. A method for determining an amount of tamoxifen and metabolites thereof in a human sample in a single mass spectrometry assay, comprising: (a) ionizing said tamoxifen and metabolites to produce one or more ions detectable by mass spectrometry, wherein said tamoxifen metabolites comprise norendoxifen; (b) detecting the amount of the ion(s) from step (a) by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of each of tamoxifen and metabolites in said sample, wherein the method has a limit of quantitation less than or equal to 5 ng/mL of norendoxifen.
 14. The method of claim 13, further comprising protein precipitation prior to step (a).
 15. The method of claim 13, further comprising purification prior to step (a).
 16. The method of claim 15, wherein said purification comprises filtration.
 17. The method of claim 15, wherein said purification comprises liquid chromatography.
 18. The method of claim 17, wherein said liquid chromatography is high pressure liquid chromatography (HPLC).
 19. The method of claim 13, further comprising detecting the amount of an internal standard.
 20. The method of claim 19, wherein said internal standard is a deuterated internal standard.
 21. The method of claim 13, wherein said ionization is by atmospheric pressure chemical ionization (APCI).
 22. The method of claim 13, wherein said ionization is in positive ion mode.
 23. The method of claim 13, wherein said sample is a serum sample.
 24. The method of claim 13, wherein said mass spectrometry is tandem mass spectrometry.
 25. The method of claim 13, wherein said metabolites comprise endoxifen or N-Desmethyl-4-Hydroxy Tamoxifen.
 26. The method of claim 13, wherein said metabolites comprise 4′-Hydroxy Tamoxifen.
 27. The method of claim 13, wherein said metabolites comprise 4-Hydroxy Tamoxifen.
 28. The method of claim 13, wherein said metabolites comprise N-Desmethyl-4′-Hydroxy Tamoxifen.
 29. The method of claim 13, wherein said metabolites comprise N-Desmethyl Tamoxifen.
 30. The method of claim 13, wherein said metabolites comprise norendoxifen, endoxifen, 4′-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4′-Hydroxy Tamoxifen, and N-Desmethyl-4-Hydroxy Tamoxifen. 